<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml">
<head>
  <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
  <meta http-equiv="Content-Style-Type" content="text/css" />
  <meta name="generator" content="pandoc" />
  <title></title>
  <style type="text/css">code{white-space: pre;}</style>
  <link rel="stylesheet" href="./vendor/bd.css" type="text/css" />
</head>
<body>
<div id="TOC">
<ul>
<li><a href="#chapter-3-oxidative-degradation">CHAPTER 3 Oxidative Degradation</a></li>
<li><a href="#第三章-氧化降解">第三章 氧化降解</a><ul>
<li><a href="#introduction">3.1 Introduction</a></li>
<li><a href="#前言">3.1 前言</a></li>
<li><a href="#free-radical-mediated-autooxidation">3.2 Free Radical-mediated Autooxidation</a></li>
<li><a href="#自由基介导的自然氧化">3.2 自由基介导的自然氧化</a><ul>
<li><a href="#origin-of-free-radicals-fenton-reaction-and-udenfriend-reaction">3.2.1 Origin of Free Radicals: Fenton Reaction and Udenfriend Reaction</a></li>
<li><a href="#自由基的来源芬顿反应和-udenfriend-反应">3.2.1 自由基的来源：芬顿反应和 Udenfriend 反应</a></li>
<li><a href="#origin-of-free-radicals-homolytic-cleavage-of-peroxides-by-thermolysis-and-heterolytic-cleavage-of-peroxides-by-metal-ion-oxidation">3.2.2 Origin of Free Radicals: Homolytic Cleavage of Peroxides by Thermolysis and Heterolytic Cleavage of Peroxides by Metal Ion Oxidation</a></li>
<li><a href="#自由基的来源过氧化物热解均裂或金属离子氧化过氧化物而异裂">3.2.2 自由基的来源：过氧化物热解均裂或金属离子氧化过氧化物而异裂</a></li>
<li><a href="#autooxidative-radical-chain-reactions-and-their-kinetic-behavior">3.2.3 Autooxidative Radical Chain Reactions and Their Kinetic Behavior</a></li>
<li><a href="#自然氧化中的自由基链式反应极其动力学行为">3.2.3 自然氧化中的自由基链式反应极其动力学行为</a></li>
<li><a href="#additional-reactions-of-free-radicals">3.2.4 Additional Reactions of Free Radicals</a></li>
<li><a href="#自由基加成反应">3.2.4 自由基加成反应</a></li>
</ul></li>
<li><a href="#non-radical-reactions-of-peroxides">3.3 Non-radical Reactions of Peroxides</a></li>
<li><a href="#过氧化物的非自由基反应">3.3 过氧化物的非自由基反应</a><ul>
<li><a href="#heterolytic-cleavage-of-peroxides-and-oxidation-of-amines-sulfides-and-related-species">3.3.1 Heterolytic Cleavage of Peroxides and Oxidation of Amines, Sulfides, and Related Species</a></li>
<li><a href="#过氧化物的异裂与胺硫化物极其相关物类的氧化">3.3.1 过氧化物的异裂与胺、硫化物极其相关物类的氧化</a></li>
<li><a href="#heterolytic-cleavage-of-peroxides-and-formation-of-epoxides">3.3.2 Heterolytic Cleavage of Peroxides and Formation of Epoxides</a></li>
<li><a href="#过氧化物的异裂与环氧化物的生成">3.3.2 过氧化物的异裂与环氧化物的生成</a></li>
</ul></li>
<li><a href="#carbanionenolate-mediated-autooxidation-base-catalyzed-autooxidation">3.4 Carbanion/enolate-mediated Autooxidation (Base-catalyzed Autooxidation)</a></li>
<li><a href="#碳负离子烯醇负离子介导的自然氧化碱催化自然氧化">3.4 碳负离子/烯醇负离子介导的自然氧化（碱催化自然氧化）</a></li>
<li><a href="#oxidation-pathways-of-drugs-with-various-structures">3.5 Oxidation Pathways of Drugs with Various Structures</a></li>
<li><a href="#结构各异的药物分子的氧化途径">3.5 结构各异的药物分子的氧化途径</a><ul>
<li><a href="#allylic--and-benzylic-type-positions-susceptible-to-hydrogen-abstraction-by-free-radicals">3.5.1 Allylic- and Benzylic-type Positions Susceptible to Hydrogen Abstraction by Free Radicals</a></li>
<li><a href="#烯丙位苄位容易被自由基夺氢">3.5.1 烯丙位、苄位容易被自由基夺氢</a></li>
<li><a href="#double-bonds-susceptible-to-addition-by-hydroperoxides">3.5.2 Double Bonds Susceptible to Addition by Hydroperoxides</a></li>
<li><a href="#过氧化氢可加成双键">3.5.2 过氧化氢可加成双键</a></li>
<li><a href="#tertiary-amines">3.5.3 Tertiary Amines</a></li>
<li><a href="#叔胺">3.5.3 叔胺</a></li>
<li><a href="#primary-and-secondary-amines">3.5.4 Primary and Secondary Amines</a></li>
<li><a href="#伯胺与仲胺">3.5.4 伯胺与仲胺</a></li>
<li><a href="#enamines-and-imines-schiff-bases">3.5.5 Enamines and Imines (Schiff Bases)</a></li>
<li><a href="#烯胺和亚胺希夫碱">3.5.5 烯胺和亚胺(希夫碱)</a></li>
<li><a href="#thioethers-organic-sulfides-sulfoxides-thiols-and-related-species">3.5.6 Thioethers (Organic Sulfides), Sulfoxides, Thiols and Related Species</a></li>
<li><a href="#硫醚有机硫化物亚砜硫醇以及相关物类">3.5.6 硫醚(有机硫化物)，亚砜，硫醇以及相关物类</a></li>
<li><a href="#examples-of-carbanionenolate-mediated-autooxidation">3.5.7 Examples of Carbanion/enolate-mediated Autooxidation</a></li>
<li><a href="#碳正离子烯醇负离子介导的自然氧化的实例">3.5.7 碳正离子/烯醇负离子介导的自然氧化的实例</a></li>
<li><a href="#oxidation-of-drugs-containing-alcohol-aldehyde-and-ketone-functionalities">3.5.8 Oxidation of Drugs Containing Alcohol, Aldehyde, and Ketone Functionalities</a></li>
<li><a href="#含有醇醛酮官能团的药物分子的氧化">3.5.8 含有醇、醛、酮官能团的药物分子的氧化</a></li>
<li><a href="#oxidation-of-aromatic-rings-formation-of-phenols-polyphenols-and-quinones">3.5.9 Oxidation of Aromatic Rings: Formation of Phenols, Polyphenols, and Quinones</a></li>
<li><a href="#芳香环的氧化生成酚多酚醌">3.5.9 芳香环的氧化：生成酚、多酚、醌</a></li>
<li><a href="#oxidation-of-heterocyclic-aromatic-rings">3.5.10 Oxidation of Heterocyclic Aromatic Rings</a></li>
<li><a href="#芳香杂环的氧化">3.5.10 芳香杂环的氧化</a></li>
<li><a href="#miscellaneous-oxidative-degradations">3.5.11 Miscellaneous Oxidative Degradations</a></li>
<li><a href="#其他氧化降解">3.5.11 其他氧化降解</a></li>
</ul></li>
<li><a href="#references">References</a></li>
</ul></li>
</ul>
</div>
<table><tr><td width="59%" height="0"></td><td></td></tr>

<tr><td>

<h1 id="chapter-3-oxidative-degradation"><a href="#chapter-3-oxidative-degradation">CHAPTER 3 Oxidative Degradation</a></h1>
</td><td>

<h1 id="第三章-氧化降解"><a href="#第三章-氧化降解">第三章 氧化降解</a></h1>
</td></tr>
<tr><td>

<h2 id="introduction"><a href="#introduction">3.1 Introduction</a></h2>
</td><td>

<h2 id="前言"><a href="#前言">3.1 前言</a></h2>
</td></tr>
<tr><td>

<p>Oxidative degradation of drugs is one of the most common degradation pathways but perhaps the most complex one. In the vast majority cases, the ultimate source of the oxidizing agent is molecular oxygen (O<sub>2</sub>), which accounts for approximately 21% of the atmosphere. Because the oxidation of many organic compounds by molecular oxygen is seemingly &quot;spontaneous and uncatalyzed&quot;, this type of oxidation is usually called &quot;autooxidation&quot; or &quot;autoxidation&quot;.<span class="cite-ref"><sup>[1]</sup></span> Other terms such as &quot;aerial oxidation&quot; or &quot;allomerization&quot; are also used. The term &quot;allomerization&quot; was initially used by Willstatter and Stoll between 1911 and 1913 to describe the solution degradation of chlorophylls by exposure to molecular oxygen.<span class="cite-ref"><sup>[2,3]</sup></span> Hence, the autooxidation of chlorophylls is referred to as &quot;allomerization&quot;. Since most organic compounds are in a singlet state, that is, electron-paired, while molecular oxygen in its ground state is a triplet species, the reaction between most organic compounds and molecular oxygen is a kinetically forbidden process owing to violation of the spin conservation rule.<span class="cite-ref"><sup>[4]</sup></span> Hence, the &quot;spontaneous&quot; autooxidation reaction usually involves activation of ground state molecular oxygen, during which process the latter can be activated into a few species of various reactivity such as superoxide anion radical (O<sub>2</sub><sup>-•</sup>), hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), hydroxyl free radical (HO<sup>•</sup>), and singlet oxygen (<sup>1</sup>O<sub>2</sub>). Collectively, these species are usually called &quot;reactive oxygen species&quot; or &quot;ROS&quot;.<span class="cite-ref"><sup>[5]</sup></span> Redox-active transition metal ions, most commonly iron and copper ions, usually play a key catalytic role in the activation process that produces O<sub>2</sub><sup>-•</sup>, H<sub>2</sub>O<sub>2</sub>, and HO<sup>•</sup>. This process, involving electron transfer and free radicals, is the most significant one in autooxidation of drugs. On the other hand, ozone, typically formed by electric sparking or vacuum UV irradiation, is usually not a concern for the oxidative degradation of drugs. Singlet oxygen, usually generated under photo-sensitization conditions, plays an important role in photooxidative degradation of drugs, which will be discussed in Chapter 6.</p>
</td><td>

<p>氧化降解是药物降解途径中最常见的机理，同时也是最复杂。在大多数实例中，空气中体积比占 21% 之多的氧气是最主要的氧化剂。鉴于许多有机物与氧气的反应都是无需催化剂且自发进行的，此类氧化反应被称为自然氧化(autoxidation、autooxidation、aerial oxidation、allomerization)。<span class="cite-ref"><sup>[1]</sup></span> 1911年至1913年期间，Willstatter 和 Stoll 最早使用了 allomerization 这一术语，用以描述叶绿素溶液在氧气存在条件下的降解行为。<span class="cite-ref"><sup>[2,3]</sup></span> 其研究中即以 allomerization 代指叶绿素的自然氧化。一般而言，大多数有机物都处于单线态，即电子成对状态，基态为三线态的氧分子与基态为单线态的有机分子之间的反应是自旋禁阻的，故而无动力学优势。<span class="cite-ref"><sup>[4]</sup></span> 因此，参与自发进行的自然氧化必然涉及基态的氧分子的激发，这将产生活性氧类(Reactive oxygen species, ROS)，包括超氧阴离子自由基(O<sub>2</sub><sup>-•</sup>)、过氧化氢(H<sub>2</sub>O<sub>2</sub>)、羟自由基(HO<sup>•</sup>)、单线激发态氧(<sup>1</sup>O<sub>2</sub>)等。<span class="cite-ref"><sup>[5]</sup></span> 具有氧化还原活性的过渡金属，一般是铁或铜的离子，往往在活性氧类的生成中扮演重要角色。此过程涉及电子转移和自由基反应，这对药物的自然氧化意义重大。另一方面，火花放电或高能紫外线激发能产生臭氧，但一般与药物的氧化降解关系不大。光敏化条件下产生的单线态氧，是光氧化降解的主角，将在第六章详细论述。</p>
</td></tr>
<tr><td>

<p>Certain electron-rich species, like many phenol or polyphenol type compounds, seem to be capable of reacting with molecular oxygen without apparent activation of the latter; one example is tetrachlorohydroquinone (TCHQ), a metabolite of pentachlorophenol (PCP).<span class="cite-ref"><sup>[6]</sup></span> Nevertheless, whether the autooxidation of these compounds or any singlet organic molecules is truly without the involvement of transition metal catalysis is still debatable. For one thing, it is extremely difficult to completely remove all residual transition metal ions experimentally. Miller et al. hypothesized that &quot;true&quot; autooxidation, that is, autooxidation without redox transition metal catalysis, is negligible and the rate constant of such a true autooxidation is estimated to be ~10<sup>-5</sup> M<sup>-1</sup>s<sup>-1</sup>.<span class="cite-ref"><sup>[4]</sup></span></p>
</td><td>

<p>富电子化合物，比如苯酚、多酚可直接与氧分子反应，而无需经历氧分子的激发。比如，四氯氫醌(tetrachlorohydroquinone, TCHQ)是四氯苯酚(pentachlorophenol, PCP)的代谢物。<span class="cite-ref"><sup>[6]</sup></span> 但是，这些化合物的自然降解是否真的不涉及过渡金属催化仍然存在争议，毕竟很难保证体系中不存在过渡金属离子。Miller 等人假定“真正的（完全不涉及氧化还原活性的过度金属催化）”自然降解作用及其微弱，其速率常数估算值为 ~10<sup>-5</sup> M<sup>-1</sup>s<sup>-1</sup>。<span class="cite-ref"><sup>[4]</sup></span></p>
</td></tr>
<tr><td>

<p>Drug substances that contain somewhat &quot;acidic&quot; carbonated protons (CH<sub>n</sub>, n is typically 1 to 2) tend to undergo autooxidation via carbanion/enolate-type intermediates through deprotonation. The autooxidation of these compounds, which is also referred to as base-catalyzed autooxidation, apparently does not involve radical species and its degradation kinetics is usually much faster than a free radical-mediated autooxidation. This type of non-radical-mediated auto-oxidation is much less known for its role in drug degradation, although it can be a significant degradation pathway particularly in liquid formulations.<span class="cite-ref"><sup>[7,8]</sup></span></p>
</td><td>

<p>含有“酸性”氢（比如酮的 α-H）的化合物容易生成碳负离子/烯醇负离子中间体而发生自然氧化降解。这些化合物的自然降解也被称作是碱催化自然氧化，一般不涉及自由基过程，且反应速率远远高于自由基反应。此机理是药物降解的重要途径，尤其对液体制剂意义重大<span class="cite-ref"><sup>[7,8]</sup></span>，但知之者甚少。</p>
</td></tr>
<tr><td>

<h2 id="free-radical-mediated-autooxidation"><a href="#free-radical-mediated-autooxidation">3.2 Free Radical-mediated Autooxidation</a></h2>
</td><td>

<h2 id="自由基介导的自然氧化"><a href="#自由基介导的自然氧化">3.2 自由基介导的自然氧化</a></h2>
</td></tr>
<tr><td>

<p>Free radical-mediated autooxidation of drugs usually involves redox-active transition metal ions and/or exposure to light. The latter will be covered in Chapter 6, Photochemical Degradation. The role of the metal ions in the initiation stage of a free radical-mediated autooxidation is to act as an electron donor from its lower oxidation state (or reduced state) to molecular oxygen. The commonly encountered redox-active transition metal ion pairs are Fe(II)/ Fe(III), Cu(I)/Cu(II), Mn(I)/Mn(III), Ni(I)/Ni(IV), Pb(I)/Pb(IV), Ti(III)/Ti(IV), and Co(II)/Co(III). The most relevant redox-active transition metal ions in drug degradation are iron ions, followed perhaps by copper ions. This type of transition metal-catalyzed process which generates reactive oxygen species, in particular HO<sup>•</sup> radicals, is generally referred to as the Fenton reaction or Fenton-type reaction by a great number of researchers. Nevertheless, a closely related process, called the Udenfriend reaction, is more directly relevant in the autooxidative degradation of drugs.</p>
</td><td>

<p>自由基介导的自然氧化往往涉及有氧化还原活性的过渡金属，有时还与光照有关。后者则在第六章（光化学降解）中讨论。自由基反应的引发阶段，低氧化态的金属离子提供电子给氧分子。常见的氧化还原活性的过渡金属离子有 Fe(II)/ Fe(III)、Cu(I)/Cu(II)、Mn(I)/Mn(III)、Ni(I)/Ni(IV)、Pb(I)/Pb(IV)、Ti(III)/Ti(IV)、Co(II)/Co(III)。而在药物降解中最常出现的是铁离子，其次为铜离子。过渡金属离子催化产生活性氧类(ROS)，一般为 HO<sup>•</sup>。众多学者称之为芬顿(fenton)反应或类芬顿(Fenton-type)反应。但实际上，Udenfriend 反应才与药物的自然氧化直接相关。</p>
</td></tr>
<tr><td>

<h3 id="origin-of-free-radicals-fenton-reaction-and-udenfriend-reaction"><a href="#origin-of-free-radicals-fenton-reaction-and-udenfriend-reaction">3.2.1 Origin of Free Radicals: Fenton Reaction and Udenfriend Reaction</a></h3>
</td><td>

<h3 id="自由基的来源芬顿反应和-udenfriend-反应"><a href="#自由基的来源芬顿反应和-udenfriend-反应">3.2.1 自由基的来源：芬顿反应和 Udenfriend 反应</a></h3>
</td></tr>
<tr><td>

<p>While still a London college student in 1894, H.J.H. Fenton described the oxidation of tartaric acid in aqueous solution using a mixture of H<sub>2</sub>O<sub>2</sub> and Fe(II) salt.<span class="cite-ref"><sup>[9]</sup></span> The reaction did not receive much attention until 40 years later when Haber and Weiss suggested that HO<sup>•</sup> might be produced in the Fenton reaction as the oxidizing agent (Scheme 3.1).<span class="cite-ref"><sup>[10]</sup></span></p>
</td><td>

<p>1894 年，伦敦大学学生 H.J.H. Fenton 就记述了在 H<sub>2</sub>O<sub>2</sub> 和亚铁盐水溶液中酒石酸的氧化反应。<span class="cite-ref"><sup>[9]</sup></span> 此反应并未引起多大注意，直到 40 年后 Haber 和 Weiss 提出芬顿反应可能生成了羟基自由基作为氧化剂(Scheme 3.1)。<span class="cite-ref"><sup>[10]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.1.png" alt="Scheme 3.1  " /><p class="caption"><span class="pic-ref">Scheme 3.1</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In 1954, Sydney Udenfriend and co-workers at National Heart Institute, Bethesda, Maryland, published a study which showed that aromatic compounds could be effectively hydroxylated in an aqueous solution containing Fe(II), ascorbic acid, and ethylenediamine tetraacetic acid (EDTA) when the resulting mixture was exposed to air.<span class="cite-ref"><sup>[11]</sup></span> Udenfriend et al. also demonstrated that H<sub>2</sub>O<sub>2</sub> is a critical intermediate in the reaction. This process (the Udenfriend reaction) is illustrated in Scheme 3.2.</p>
</td><td>

<p>1954 年，国家心脏学会(National Heart Institute, Bethesda, Maryland)的 Sydney Udenfriend 及其合作者的研究显示，在芳香族化合物的水溶液中加入亚铁盐、维生素C、EDTA(乙二胺四乙酸)，并暴露于空气中时，芳香化合物可迅速水解。<span class="cite-ref"><sup>[11]</sup></span> Udenfriend 等人还证实了 H<sub>2</sub>O<sub>2</sub> 是此反应的关键中间体。此过程即命名为 Udenfriend 反应，其机理见 Scheme 3.2。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.2.png" alt="Scheme 3.2  " /><p class="caption"><span class="pic-ref">Scheme 3.2</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Multiple intermediary steps are involved in the Udenfriend reaction and one of them is likely to be the Fenton reaction which turns H<sub>2</sub>O<sub>2</sub> into HO<sup>•</sup>. It appears that Fe(II){EDTA} activates molecular oxygen by transferring an electron to it. Consequently, molecular oxygen is reduced, becoming a superoxide anion radical, while Fe(II){EDTA} is oxidized into Fe(III){EDTA}. The superoxide anion radical can then transform to hydrogen peroxide by three possible routes: (1) by abstracting an H<sup>•</sup> radical, (2) via reduction by Fe(II){EDTA}, and (3) by disproportionation. The hydrogen peroxide formed can be dissociated into hydroxyl radicals upon further reaction with Fe(II){EDTA}, (the Fenton reaction). On the other hand, Fe(III){EDTA} can be recycled back to the catalytically active Fe(II){EDTA} through reduction by ascorbic acid. All the plausible steps of the Udenfriend reaction are shown in Scheme 3.3.</p>
</td><td>

<p>Udenfriend 反应涉及多步中间反应，其中一步 H2O2 转化为 HO<sup>•</sup>，类似于芬顿反应。Fe(II){EDTA} 传递电子给氧分子，从而使之活化。氧分子得到电子被还原为超氧阴离子自由基，Fe(II){EDTA} 则失去电子被氧化为 Fe(III){EDTA}。超氧阴离子自由基可通过以下三种途径转化为过氧化氢：(1) 夺取 H<sup>•</sup> 自由基，(2) 被 Fe(II){EDTA} 还原，(3) 自身歧化反应。过氧化氢与 Fe(II){EDTA} 反应则产生羟基自由基（芬顿反应）。同时，Fe(III){EDTA} 被维生素C还原为 Fe(II){EDTA}，从而构成催化循环。Udenfriend 反应的所有可能历程见 Scheme 3.3。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.3.png" alt="Scheme 3.3  " /><p class="caption"><span class="pic-ref">Scheme 3.3</span>  </p>
</div>
</td></tr>
<tr><td>

<p>As shown in Scheme 3.3, the Fenton reaction can be considered an important step in the multiple-step Udenfriend reaction. In order for both the Fenton reaction and Udenfriend reaction to be fully operative under near neutral pH conditions, a good iron chelating agent such as EDTA is needed to prevent iron ions, in particular Fe(III), from precipitating out of solution, especially at pH approaching neutral. A consequence of the use of a chelating agent is that it could lower the reduction potential (E°) of Fe(III)/Fe(II), depending upon the nature of the chelator. For example, E°' for Fe(III)/Fe(II) at pH 7.0 is 0.11 V, while E°' for Fe(III){ferrioxamine}/Fe(II){ferrooxamine} is -0.45 V.<span class="cite-ref"><sup>[12]</sup></span> On the other hand, E°' for Fe(III){EDTA}/Fe(II){EDTA} at pH 7.0 is 0.12 V, which is essentially the same as E°' for the non-chelated Fe(III)/Fe(II).<span class="cite-ref"><sup>[13]</sup></span> Please note that the frequently quoted standard reduction potential (E°) value of 0.77 V for Fe(III)/Fe(II) is obtained under the &quot;standard&quot; condition in which the pH is 0 (the standard concentration of H<sup>+</sup> is 1M).<span class="cite-ref"><sup>[14]</sup></span> Owing to the low reduction potential (E°') and much improved solubility at pH 7.0 for EDTA chelated Fe(III)/Fe(II), EDTA greatly facilitates the Fenton and Udenfriend reactions because the soluble Fe(II)EDTA should be a much more efficient electron donor to molecular oxygen at neutral pH. in the experiments carried out by Udenfriend et al., the use of EDTA markedly enhanced the rate of the autooxidation reactions.</p>
</td><td>

<p>如 Scheme 3.3 所示，芬顿反应可看做是 Udenfriend 反应的中间一环。为了让芬顿反应和 Udenfriend 反应能在中性 pH 中进行，需加入螯合剂，如 EDTA ，以防止铁离子因水解而沉降。且加入螯合剂能降低 Fe(III)/Fe(II) 的还原电势(E°)。例如，pH 7.0 时，Fe(III)/Fe(II) 的 E°' 为 0.11 V；Fe(III){ferrioxamine}/Fe(II){ferrooxamine} 则为 -0.45 V。<span class="cite-ref"><sup>[12]</sup></span> 但 Fe(III){EDTA}/Fe(II){EDTA} 的E°' 为 0.12 V，与未螯合的 Fe(III)/Fe(II) 接近。<span class="cite-ref"><sup>[13]</sup></span> 但请读者注意，Fe(III)/Fe(II) 的标准电极电势 E° 为 0.77 V，这是在标准状况下所测得的，此时的 pH 为 0 （H<sup>+</sup> 浓度为 1 M）。<span class="cite-ref"><sup>[14]</sup></span> pH 7.0 时，EDTA 能降低还原电势且明显提高铁盐的溶解度，这使得芬顿反应或 Udenfriend 反应更容易进行。这是因为，中性pH 下 Fe(II){EDTA} 能高效地传递电子给氧分子。在 Udenfriend 等人的实验中，EDTA 能够明显提高反应速度。</p>
</td></tr>
<tr><td>

<p>Note that the steps shown in Scheme 3.3 are probably simplified working models for both the Fenton and Udenfriend reactions and the thermodynamic feasibility of the reaction sequence is demonstrated by the use of the standard reduction potential, E°, rather than the reduction potential at neutral pH, E°'. A great deal of effort has been put into studying the detailed mechanism of the Fenton reaction over the past few decades.<span class="cite-ref"><sup>[15-17]</sup></span> One of the key questions, which is still debatable today, has been whether a HO<sup>•</sup> free radical is really produced in the Fenton reaction.<span class="cite-ref"><sup>[18]</sup></span> The alternate hypothesis for the oxidation intermediate is a ferryl species either in the form of a Fe(IV)O<sup>2+</sup> ion or a Fe(IV)O<sup>+•</sup> radical cation. The Fe(IV)O<sup>2+</sup> ion has been generated and characterized by a number of techniques.<span class="cite-ref"><sup>[19,20]</sup></span> Based on the discovery of some chemistry that is unique to the Fe(IV)O<sup>2+</sup> ion, such as oxygen atom transfer to sulfoxides, its involvement in the Fenton reaction was ruled out.<span class="cite-ref"><sup>[21]</sup></span> With regard to the Fe(IV)O<sup>+•</sup> radical cation, although its hypothesized presence as the critical oxidizing intermediate in oxidative enzymes such as cytochrome P450<span class="cite-ref"><sup>[22]</sup></span> has been recently verified experimentally,<span class="cite-ref"><sup>[23]</sup></span> the possibility of its being the intermediate in the regular Fenton reaction seems still very low. in order to stabilize a high valence iron species like the Fe(IV)O<sup>+•</sup> radical cation, strong, electron-rich ligands such as porphyrins are required. Hence, in the regular Fenton and related Udenfriend reactions, where the ligands are typically not as strong or electron-rich as porphyrins, HO<sup>•</sup> radical is most likely to be the oxidizing intermediate. Obviously, nobody believes that this HO<sup>•</sup> radical would behave like one that is generated by γ-radiation of water. The HO<sup>•</sup> radical intermediate in the regular Fenton and related Udenfriend reactions is most probably formed in a site-specific manner.<span class="cite-ref"><sup>[24-26]</sup></span> Such a HO<sup>•</sup> radical would not diffuse or react too far away from the point of its formation. In addition, the site-specific HO<sup>•</sup> radical displays muted reactivity compared to that generated by γ-radiation.</p>
</td><td>

<p>Scheme 3.3 中的一系列反应可看做是 Fenton 反应和 Udenfriend 反应的简化模型，此处使用标准还原电势 E°（而非中性 pH 时的还原电势 E°'）说明了这些反应是热力学可行的。过去的几十年里，人们花费了很大精力来研究 Fenton 反应的详细机理。<span class="cite-ref"><sup>[15-17]</sup></span> 但 Fenton 反应中是否真的产生了 HO<sup>•</sup> 自由基呢？此问题至今仍有争议。<span class="cite-ref"><sup>[18]</sup></span> 另外一种假说认为 Fenton 反应中生成的强氧化性中间体是高价铁的复合物：Fe(IV)O<sup>2+</sup> 正离子或 Fe(IV)O<sup>+•</sup> 自由基正离子。现在有一些手段可以产生并表征 Fe(IV)O<sup>2+</sup> 离子。<span class="cite-ref"><sup>[19,20]</sup></span> 在 Fenton 体系中却没有发现 Fe(IV)O<sup>2+</sup> 的一些特有反应（比如把氧原子转移给亚砜而生成砜）。<span class="cite-ref"><sup>[21]</sup></span> 至于说 Fe(IV)O<sup>+•</sup> 自由基正离子，虽然已经证实它是某些氧化酶（比如细胞色素 P450）的关键氧化性中间体<span class="cite-ref"><sup>[23]</sup></span>，但常规的 Fenton 反应能产生 Fe(IV)O<sup>+•</sup>，其可能性毕竟很低。为了使高价态铁离子（如 Fe(IV)O<sup>+•</sup>）稳定存在，需要使用富电子的强配体（比如卟啉）。然而，常规的 Fenton 反应和 Udenfriend 反应中使用的配体远远达不到卟啉的水平，更有可能是生成了 HO<sup>•</sup> 自由基作为氧化性中间体。显然，没人愿意相信此 HO<sup>•</sup> 自由基会像γ射线照射水所产生的羟基自由基那样反应。常规的 Fenton 反应和相关 Udenfriend 反应中，羟基自由基是特定区域生成的。<span class="cite-ref"><sup>[24-26]</sup></span> 此自由基不怎么扩散，而只会在生成位点附近发生反应。此外，相比于γ射线照射所形成的 HO<sup>•</sup> ，这些区域特异的 HO<sup>•</sup> 表现出更低的反应活性。</p>
</td></tr>
<tr><td>

<p>As illustrated above, the Udenfriend reaction consists of three key components, that is, a transition redox metal ion (Fe<sup>2+</sup>), a good chelating agent (EDTA), and a reducing agent (ascorbic acid). In other words, the combination of these three types of components would effectively convert molecular oxygen into a few reactive oxygen species (ROS) including H<sub>2</sub>O<sub>2</sub> and HO<sup>•</sup> radicals. Indeed, studies have shown that other transition metal ions, chelating agents and reducing agents/antioxidants could replace Fe(II), EDTA, and ascorbic acid, respectively, in the Udenfriend reaction. For example, in several mechanistic studies where hydroxylation of aromatic compounds was used as the indicator for HO<sup>•</sup> formation or DNA damage under oxidative stress, it has been demonstrated that a number of transition redox metal ions, such as Cu(I), can replace Fe(II).<span class="cite-ref"><sup>[27,28]</sup></span> On the other hand, several metal chelators, such as citrate<span class="cite-ref"><sup>[29]</sup></span> and diethylenetriamine pentaacetic acid (DTPA) (also called DETA-PAC),<span class="cite-ref"><sup>[30]</sup></span> can substitute for EDTA. This is consistent with the fact that the reduction potentials of the DTPA chelated and citrate chelated iron pairs are 0.165 V , and ~ 0.1V, respectively, which are similar to the reduction potential (0.12 V) of an EDTA chelated iron pair. These studies, along with that of Kasai and Nishimura,<span class="cite-ref"><sup>[28]</sup></span> also implied that several reducing agents, like derivatives of phenol (e.g. trolox, a vitamin E analog)<span class="cite-ref"><sup>[34]</sup></span> and catechol,<span class="cite-ref"><sup>[35]</sup></span> are capable of recycling Fe(III) back to Fe(II), suggesting that they can take the role of ascorbic acid in the Udenfriend reaction. Among all of the species implicated in the above studies as replacements for the three key components of the Udenfriend reaction, those that are pharmaceutically and/or physiologically relevant are summarized in Table 3.1.</p>
</td><td>

<p>如前文所述，Udenfriend 反应需要三个关键组分：氧化还原活性过渡金属离子（Fe<sup>2+</sup>）、络合剂（EDTA）和还原剂（维生素C）。换言之，这三种成分组合在一起可高效地将氧分子转化为活性氧类，其中包括 H<sub>2</sub>O<sub>2</sub> 和 HO<sup>•</sup> 自由基。实际上，有研究表明，其他过渡金属离子、络合剂和还原剂/抗氧化剂也能充当相应的角色，从而构成 Udenfriend 反应。例如，使用芳香化合物作为 HO<sup>•</sup> 指示剂，研究羟基化反应的机理，实验发现其他的一些过渡金属离子，比如 Cu(I)，可代替 Fe(II)；且在研究氧化应激对 DNA 损伤时也观测到了相同的现象。<span class="cite-ref"><sup>[27,28]</sup></span> 另一方面，其他的金属络合剂，比如柠檬酸盐<span class="cite-ref"><sup>[29]</sup></span>和二乙基三胺五乙酸(diethylenetriamine pentaacetic acid，简称 DTPA 或 DETA-PAC)<span class="cite-ref"><sup>[30]</sup></span>可替代 EDTA。这从络合离子的还原电势即可看出：DTPA 和柠檬酸盐络合的铁离子的还原电势分别为 0.165 V 和 ~ 0.1V；这与 EDTA 络合的铁离子接近（0.12 V）。上述研究以及 Kasai 和 Nishimura 的实验结果<span class="cite-ref"><sup>[28]</sup></span>都显示了其他还原剂，如酚类衍生物（例如 trolox，一个类似于维生素E 的分子）<span class="cite-ref"><sup>[34]</sup></span>和儿茶酚<span class="cite-ref"><sup>[35]</sup></span>，同样可以将 Fe(III) 还原回 Fe(II)，这意味着它们可以取代维生素C 在 Udenfriend 反应中的角色。上述研究中发现了多种可充当 Udenfriend 反应组分的物质，在药学或生物学中可能涉及的那些已列于 表 3.1。</p>
</td></tr>
<tr><td colspan=2>
<div class="figure">
<img src="png/21.png" />
</div>
</td></tr>
<tr><td>

<p>Since chelating agents and antioxidants are frequently used in the formulation of drug products for the purpose of product preservation and stability, the Udenfriend reaction has a direct impact on the stability of a drug product formulated with a combination of a chelating agent and an antioxidant (not necessarily limited to those listed in Table 3.1). Such a drug product could potentially be intrinsically vulnerable to autooxidation, because a slight increase of a transition redox metal ion, either from the primary packaging, raw materials or during manufacturing, into the formulation could trigger the Udenfriend process, causing decreased stability of the finished drug product. Nevertheless, this does not mean that any combination of the three components from each of the three categories in Table 3.1 above would automatically constitute a Udenfriend reaction system, because the thermodynamics and/or kinetics of such a combination may not always be favorable for the reaction to proceed.</p>
</td><td>

<p>药物制剂中常常会使用螯合剂和抗氧化剂来保证产品容易保存或提高产品的稳定性。但当制剂产品中同时含有螯合剂和抗氧化剂（不仅限于 表 3.1 中列出的那些）时，Udenfriend 反应可明显影响其稳定性。此时，制剂产品可能会变得对自然氧化非常敏感乃至十分脆弱，因为氧化还原活性的过渡金属离子的含量仅有轻微增加时（可能来自于主包装、原材料或在生产过程中引入）都有可能触发 Udenfriend 反应，导致产品的稳定性降低。但是，这并不是说任意组合 表 3.1 中的三种组分就可以构成 Udenfriend 反应体系，因为一些组合可能会因为缺少热力学或动力学优势而难以发生反应。</p>
</td></tr>
<tr><td>

<p>Sometimes, the drug molecule itself can be the chelating agent for redox transition metal ions. As a result, the drug may be oxidized at a particular site by the ROS formed nearby. in such cases, use of additional chelating agent such as EDTA can inhibit the oxidation occurring at that particular site. Nevertheless, the drug may be oxidized at yet another site or sites by a new Udenfriend system that now consists of EDTA that replaces the drug molecule.</p>
</td><td>

<p>有时，药物分子也可能成为氧化还原活性的过渡金属离子的络合剂。结果，药物分子的特定位点将被附近生成活性氧类氧化。此时，在体系中加入 EDTA 等络合剂反而能抑制此特定位点的氧化。但是 EDTA 取代了原本的药物分子，这依然是一个 Udenfriend 体系，药物分子的其他位点还会有可能被氧化。</p>
</td></tr>
<tr><td>

<h3 id="origin-of-free-radicals-homolytic-cleavage-of-peroxides-by-thermolysis-and-heterolytic-cleavage-of-peroxides-by-metal-ion-oxidation"><a href="#origin-of-free-radicals-homolytic-cleavage-of-peroxides-by-thermolysis-and-heterolytic-cleavage-of-peroxides-by-metal-ion-oxidation">3.2.2 Origin of Free Radicals: Homolytic Cleavage of Peroxides by Thermolysis and Heterolytic Cleavage of Peroxides by Metal Ion Oxidation</a></h3>
</td><td>

<h3 id="自由基的来源过氧化物热解均裂或金属离子氧化过氧化物而异裂"><a href="#自由基的来源过氧化物热解均裂或金属离子氧化过氧化物而异裂">3.2.2 自由基的来源：过氧化物热解均裂或金属离子氧化过氧化物而异裂</a></h3>
</td></tr>
<tr><td>

<p>As shown in Section 3.2.1, hydrogen peroxide is generated during the activation of molecular oxygen in the Udenfriend reaction. Certain polymeric excipients are prone to autooxidation leading to the formation of peroxides. For example, it was reported that pharmaceutical grade polyethylene glycol (PEG) and povidone contain various levels of peroxides including hydrogen peroxide.<span class="cite-ref"><sup>[39-41]</sup></span> The O-O bond of the peroxide is weak and susceptible to thermal decomposition, in addition to the transition metal ion-catalyzed cleavage (e.g. the Fenton reaction). Of the various degradation pathways of organic peroxides under thermolysis, which was reviewed by Antonovskii and Khursan,<span class="cite-ref"><sup>[42]</sup></span> homolytic cleavage of the O-O bond is a main pathway (Scheme 3.4).</p>
</td><td>

<p>小节 3.2.1 中已经展示了 Udenfriend 反应对氧分子的活化可产生过氧化氢。某些高分子赋形剂容易发生自然氧化而形成效率的过氧化物。例如，有报道称药用级聚乙二醇(PEG)和聚维酮(PVP)中含有不同水平的过氧化物，过氧化氢也在其中。<span class="cite-ref"><sup>[39-41]</sup></span> 过氧化物的 O-O 键较弱，容易受热分解或受过渡金属离子催化而断裂（例如 Fenton 反应）。由 Antonovskii 和 Khursan 的综述<span class="cite-ref"><sup>[42]</sup></span>可知，有机过氧化物热解时的降解途径主要是 O-O 键的均裂(Scheme 3.4)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.4.png" alt="Scheme 3.4  " /><p class="caption"><span class="pic-ref">Scheme 3.4</span>  </p>
</div>
</td></tr>
<tr><td>

<p>On the other hand, the oxidative state of certain metal ions such as Fe(III) and Mn(III) are capable of oxidizing hydroperoxides (ROOH) into peroxy radicals (ROO<sup>•</sup>) (Scheme 3.5)<span class="cite-ref"><sup>[43]</sup></span> owing to their strong oxidation capability as evidenced by the relatively high reduction potentials (E°) for the two metal ion pairs: Fe(III)/Fe(II), 0.77 v; Mn(III)/Mn(II) 1.5 v.<span class="cite-ref"><sup>[44]</sup></span></p>
</td><td>

<p>另一方面，高氧化态的某些金属离子，比如 Fe(III) 和 Mn(III) ，可将氢过氧化物(ROOH)氧化为过氧自由基(ROO<sup>•</sup>)，详见Scheme 3.5。<span class="cite-ref"><sup>[43]</sup></span> 此两种离子的强氧化能力的证据即相对较高的还原电势：Fe(III)/Fe(II) 0.77 v；Mn(III)/Mn(II) 1.5 v。<span class="cite-ref"><sup>[44]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.5.png" alt="Scheme 3.5  " /><p class="caption"><span class="pic-ref">Scheme 3.5</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Since the EDTA-complexed Fe(III)/Fe(II) pair has a much lower E° (0.12 v), Fe(III){EDTA} would not be expected to oxidize hydroperoxides effectively into the corresponding alkylperoxyl radicals anymore near neutral pH, because the E° of a typical alkylperoxyl radical is in the range 0.77-1.44 v,<span class="cite-ref"><sup>[45]</sup></span> resulting in a thermodynamically unfavorable positive ΔG value. On the other hand, Fe(II){EDTA} is capable of decomposing ROOH to RO<sup>•</sup> (Scheme 3.6) in a way similar to the Fenton reaction.</p>
</td><td>

<p>EDTA 络合的 Fe(III)/Fe(II) 具有更低的 E° (0.12 v)，而典型烷基过氧自由基的 E° 约为0.77-1.44 v<span class="cite-ref"><sup>[45]</sup></span>，因此 Fe(III){EDTA} 在中性 pH 下无法有效地将烷基氢过氧化物氧化为相应的烷基过氧自由基（此反应的 ΔG 为正值，无热力学优势）。另一方面，Fe(II){EDTA} 可分解 ROOH 生成 RO<sup>•</sup>，类似于 Fenton 反应(Scheme 3.6)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.6.png" alt="Scheme 3.6  " /><p class="caption"><span class="pic-ref">Scheme 3.6</span>  </p>
</div>
</td></tr>
<tr><td>

<h3 id="autooxidative-radical-chain-reactions-and-their-kinetic-behavior"><a href="#autooxidative-radical-chain-reactions-and-their-kinetic-behavior">3.2.3 Autooxidative Radical Chain Reactions and Their Kinetic Behavior</a></h3>
</td><td>

<h3 id="自然氧化中的自由基链式反应极其动力学行为"><a href="#自然氧化中的自由基链式反应极其动力学行为">3.2.3 自然氧化中的自由基链式反应极其动力学行为</a></h3>
</td></tr>
<tr><td>

<p>As discussed above, various oxygen-based free radicals can be formed during the Fenton reaction, Udenfriend reaction, and decomposition of peroxides and hydroperoxides. Once these radicals are formed, they can trigger a chain reaction which consists of the following three stages: initiation, propagation, and termination. The following scheme (Scheme 3.7) uses peroxyl radicals (XOO<sup>•</sup> , X = alkyl, H) as representative oxygen-based free radical initiators.</p>
</td><td>

<p>前文已述，Fenton 反应、Udenfriend 反应以及过氧化物和氢过氧化物的分解可产生氧自由基。这些自由基一旦形成，便可引发链式反应，这由三个阶段构成：链引发、链增长、链终止。Scheme 3.7 以典型的氧自由基——过氧自由基(XOO<sup>•</sup> , X = 烷基, H)为例，展示了一个完整的链式反应。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.7.png" alt="Scheme 3.7  " /><p class="caption"><span class="pic-ref">Scheme 3.7</span>  </p>
</div>
</td></tr>
<tr><td>

<p>in Scheme 3.7, RH represents any species that can donate an H<sup>•</sup> which includes the oxidation substrate. in autooxidation, the initiation stage is usually a slow process, which can be impacted by a number of factors such as temperature, pH, moisture level (in solid state autooxidation), and low levels of impurities, in particular trace levels of transition metal ions. in pharmaceutical products, some components or impurities of the components can inhibit (or slow down) the autooxidation process. Because of these factors, radical-mediated autooxidation displays a variable induction period, during which time no significant oxidation is observed. During the propagation stage, the chain reaction is sustained by continuous generation of ROO<sup>•</sup> and R<sup>•</sup> radicals at the expense of consuming the oxidation substrates (RH) and molecular oxygen. The reaction between R<sup>•</sup> and O<sub>2</sub> (Step 2 in Scheme 3.7) is diffusion-controlled (i.e. the rate constant k is ~ 10<sup>9</sup> M<sup>-1</sup> s<sup>-1</sup>),<span class="cite-ref"><sup>[46]</sup></span> while the rate constant of an allylic H abstract reaction by ROO<sup>•</sup> is typically in the range of ~ 0.1-60 M<sup>-1</sup>s<sup>-1</sup>.<span class="cite-ref"><sup>[47]</sup></span> in the final termination stage, when enough radical species are present, combination of any two radicals can contribute to the termination of the chain reaction. The last step shown in the chain reaction illustrated in Scheme 3.7 is known as the Russell mechanism,<span class="cite-ref"><sup>[48]</sup></span> giving rise to ketone/aldehyde, alcohol, and singlet oxygen. it should be noted that the Russell mechanism is not the only pathway to generate the ketone/aldehyde and alcohol. These degradants may also be formed from further degradation of the hydroperoxide (ROOH) produced in the propagation stage. For an inhibited autooxidation reaction, the rate of the oxidation can be described as follows.<span class="cite-ref"><sup>[49]</sup></span></p>
</td><td>

<p>Scheme 3.7 中，RH 可以是任何能提供 H<sup>•</sup> 的分子，也可能是氧化反应的底物。在自然氧化中，链引发往往非常缓慢，且受到多种因素影响，比如温度、pH、湿度（固态的自然氧化中）、杂质特别是痕量的过渡金属离子。制剂产品中，某些组分或其中的杂质可抑制（或减慢）自然氧化进程。因此，自由基介导的自然氧化往往会有不同长短的诱发期，此间不会观测到明显的氧化产物。链增长阶段将消耗底物(RH)和氧分子，持续产生 ROO<sup>•</sup> 和 R<sup>•</sup>。R<sup>•</sup> 与 O<sub>2</sub> 的反应(Scheme 3.7 第二步)速率是扩散控制的（速率常数 k 约为 10<sup>9</sup> M<sup>-1</sup> s<sup>-1</sup> <span class="cite-ref"><sup>[46]</sup></span>）；而 ROO<sup>•</sup> 夺取烯丙位氢的反应速率常数仅为 ~ 0.1-60 M<sup>-1</sup>s<sup>-1</sup> <span class="cite-ref"><sup>[47]</sup></span>。在链终止阶段，体系中已然存在相当多的自由基，双基终止成为主要的链终止方式。Scheme 3.7 中所示的最后一个反应名为 Russell 反应<span class="cite-ref"><sup>[48]</sup></span>，可生成醛/酮、醇和单线态氧。但应当注意，Russell 反应并非生成醛/酮和醇的唯一途径。这些降解产物也可以由链增长阶段生成氢过氧化物(ROOH)进一步氧化而得到。受抑制的自然氧化反应的速率可以用下述公式描述。<span class="cite-ref"><sup>[49]</sup></span></p>
</td></tr>
<tr><td colspan=2>
<div class="figure">
<img src="png/9.png" />
</div>
</td></tr>
<tr><td>

<p>where k<sub>3</sub> is the rate constant of the propagation stage, k<sub>5</sub> is the rate constant of the inhibition reaction (R<sup>•</sup> + Inhibitor → RH + relatively stable inhibitor radical), Ri is the rate of chain initiation, and n is the stoichiometric factor of the inhibitor (antioxidant).</p>
</td><td>

<p>此处 k<sub>3</sub> 是链增长阶段的速率常数；k<sub>5</sub> 是抑制反应（R<sup>•</sup> + Inhibitor → RH + 相对稳定的自由基，Inhibitor——抑制剂）的速率常数。Ri 是链引发的速率常数，n 是抑制剂(抗氧化剂)的化学计量数。</p>
</td></tr>
<tr><td>

<p>According to this equation, the rate of an inhibited autooxidation is proportional to the concentration of the oxidation substrate, [RH], and inversely proportional to the concentration of the inhibitor, [inhibitor]. The inhibitor is usually an antioxidant in a pharmaceutical formulation. This equation also indicates that the oxygen partial pressure has no impact on the rate of autooxidation. A practical implication of this conclusion is that reducing the oxygen concentration in a pharmaceutical formulation will not usually slow down the rate of the free radical-mediated autooxidation, unless oxygen can be almost completely removed from the formulation. The autooxidation kinetic study performed by Burton and Ingold with styrene as the oxidation substrate and vitamin E as the antioxidant showed a clear induction period, followed by a rapid surge of oxidation when the antioxidant was consumed;<span class="cite-ref"><sup>[49]</sup></span> an illustrative plot is shown in Figure 3.1 based on their work, which resembles the kinetic behavior of oxidizable drugs in autooxidation where generation of radicals is rate limiting.<span class="cite-ref"><sup>[50]</sup></span></p>
</td><td>

<p>根据此方程，受抑制的自然氧化反应的速率与底物的浓度 [RH] 成正比，与抑制剂的浓度 [Inhibitor] 成反比。抑制剂一般是制剂中的抗氧化剂。此方程还说明氧气的分压不影响自然氧化反应的速率。这意味着降低制剂产品中的氧气浓度并不能降低自由基介导的自然氧化反应的速度，除非近乎完全地除去氧气。Burton 和 Ingold 对苯乙烯和维生素E 进行了自然氧化反应动力学研究，存在一个明显的诱发期，当抗氧化剂被消耗干净后，迅速生成大量氧化产物。<span class="cite-ref"><sup>[49]</sup></span> 此过程可以用 Figure 3.1 表示；当自由基的产生受到抑制时，自然氧化反应将呈现类似的动力学特征。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.1.png" alt="Figure 3.1   受抑制的自然氧化反应的动力学行为。箭头指示了抑制剂被完全消耗的时间点。 Kinetic behavior of an inhibited free radical mediated autooxidation. The arrow indicates the time point when the inhibitor was consumed." /><p class="caption"><span class="pic-ref">Figure 3.1</span>   受抑制的自然氧化反应的动力学行为。箭头指示了抑制剂被完全消耗的时间点。<br /> Kinetic behavior of an inhibited free radical mediated autooxidation. The arrow indicates the time point when the inhibitor was consumed.</p>
</div>
</td></tr>
<tr><td>

<p>In addition to the pathways involved in the three stages of a typical chain reaction shown in Scheme 3.7, the two additional pathways that were discussed in Section 3.2.2 can also occur in an autooxidative chain reaction. These two pathways relate to further decomposition of the organic peroxide and hydroperoxide, which are described below.</p>
</td><td>

<p>除了 Scheme 3.7 中所示的典型链式反应，自然氧化中还可能出现小节 3.2.2 中讨论过的两种反应途径。这两种途径涉及有机过氧化物和氢过氧化物的分解，下文将会讨论。</p>
</td></tr>
<tr><td>

<p>Both the peroxide and hydroperoxide can undergo homolytic cleavage, as discussed in the previous section, to give alkoxyl and hydroxyl radicals. The alkoxyl radical can then abstract an H to produce alcohol (ROH).</p>
</td><td>

<p>前文已经介绍过，过氧化物和氢过氧化物皆可发生均裂，生成烷氧自由基和羟基自由基。烷氧自由基可夺氢生成醇(ROH)。</p>
</td></tr>
<tr><td>

<p>The hydroperoxide can also be oxidized by certain metal ions to produce peroxyl radical. in a typical case of autooxidation, various oxygen-based free radicals are formed: O<sub>2</sub><sup>-•</sup>, HO<sup>•</sup>, ROO<sup>•</sup>, and RO<sup>•</sup>, where ROO<sup>•</sup> is predominant. The reactivity of these radicals is in the following order: HO<sup>•</sup> &gt;RO<sup>•</sup> &gt;ROO<sup>•</sup> ~ O<sub>2</sub><sup>-•</sup>/HO<sub>2</sub><sup>•</sup> based on the O-H bond cleavage energies listed in Table 3.2.</p>
</td><td>

<p>氢过氧化物可以被某些金属离子氧化生成过氧自由基。在典型的自然氧化中，可形成多种氧自由基：O<sub>2</sub><sup>-•</sup>、HO<sup>•</sup>、ROO<sup>•</sup> 和 RO<sup>•</sup>，其中 ROO<sup>•</sup> 占主导地位。根据 表 3.2 中的 O-H 键离解能数据，其反应活性可排序为：HO<sup>•</sup> &gt;RO<sup>•</sup> &gt;ROO<sup>•</sup> ~ O<sub>2</sub><sup>-•</sup>/HO<sub>2</sub><sup>•</sup>。</p>
</td></tr>
<tr><td colspan=2>
<div class="figure">
<img src="png/10.png" />
</div>
</td></tr>
<tr><td>

<p>Table 3.2 also indicates that the allylic and benzylic methylene moieties should be quite susceptible to H abstraction by the oxygen-centered radicals, compared to secondary and tertiary alkyl CH moieties, because the bond dissociation energies of the former are significantly lower than the latter.</p>
</td><td>

<p>表 3.2 还揭示了如下事实：相比于仲碳或叔碳，氧自由基可轻易的夺取烯丙位和苄位的氢，因为后者的 C-H 键离解能明显低于前者。</p>
</td></tr>
<tr><td>

<p>Owing to the characteristics and complexity of the chain reaction discussed above, autooxidative degradation kinetics is usually difficult to replicate and predict accurately. in addition, it usually cannot be sped up by increasing temperature, because under higher temperature, homolytic cleavage of the various peroxides produced in the chain reaction, illustrated in Scheme 3.8, would become significant. This could alter the kinetic behavior, complicate the degradation pathways, and ultimately result in different degradation profiles.</p>
</td><td>

<p>鉴于链式反应的特殊性与复杂性，自然氧化反应的动力学特征往往无法重现或准确预测。此外，提高温度往往无法加快反应速度，因为更高的温度下，链式反应中生成的各种过氧化物的均裂反应(详见 Scheme 3.8)将会更加显著。这会改变体系的动力学行为，扰乱降解途径，最终导致不同的降解结果。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.8.png" alt="Scheme 3.8  " /><p class="caption"><span class="pic-ref">Scheme 3.8</span>  </p>
</div>
</td></tr>
<tr><td>

<h3 id="additional-reactions-of-free-radicals"><a href="#additional-reactions-of-free-radicals">3.2.4 Additional Reactions of Free Radicals</a></h3>
</td><td>

<h3 id="自由基加成反应"><a href="#自由基加成反应">3.2.4 自由基加成反应</a></h3>
</td></tr>
<tr><td>

<p>in the previous section, two important radical reactions were mentioned: H abstraction and a radical termination reaction. Additional radical reactions that are significant in drug degradation include radical addition to a molecule containing unsaturated bonds. This addition reaction can lead to the formation of polyperoxides,<span class="cite-ref"><sup>[58]</sup></span> oligomerization and polymerization of the oxidation substrate,<span class="cite-ref"><sup>[59]</sup></span> while cleavage of the initially formed radical adduct can lead to the formation of epoxides and alkoxy radicals<span class="cite-ref"><sup>[60]</sup></span> (Scheme 3.9).</p>
</td><td>

<p>前文介绍两种自由基反应：夺氢和自由基基终止。自由基加成反应在药物降解中意义重大，对不饱和键的加成可导致生成聚过氧化物<span class="cite-ref"><sup>[58]</sup></span>和被氧化底物的齐聚和聚合<span class="cite-ref"><sup>[59]</sup></span>。而加成产物发生消除反应则可生成环氧化物或烷氧基自由基<span class="cite-ref"><sup>[60]</sup></span>，详见 Scheme 3.9。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.9.png" alt="Scheme 3.9  " /><p class="caption"><span class="pic-ref">Scheme 3.9</span>  </p>
</div>
</td></tr>
<tr><td>

<p>For a drug substance containing a benzylic moiety, its reaction with free radicals can be either on the benzylic CH through H abstraction or via addition onto the aromatic ring as shown in Scheme 3.10. The rates of both reactions are comparable based on a study using carbon-centered radicals as the attacking free radicals.<span class="cite-ref"><sup>[61]</sup></span></p>
</td><td>

<p>自由基与含苄基的药物分子的反应，既可在苄位夺氢，也可以加成芳香环(Scheme 3.10)。以碳自由基作为进攻基团时，这两种反应的反应速率基本相当。<span class="cite-ref"><sup>[61]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.10.png" alt="Scheme 3.10  " /><p class="caption"><span class="pic-ref">Scheme 3.10</span>  </p>
</div>
</td></tr>
<tr><td>

<h2 id="non-radical-reactions-of-peroxides"><a href="#non-radical-reactions-of-peroxides">3.3 Non-radical Reactions of Peroxides</a></h2>
</td><td>

<h2 id="过氧化物的非自由基反应"><a href="#过氧化物的非自由基反应">3.3 过氧化物的非自由基反应</a></h2>
</td></tr>
<tr><td>

<h3 id="heterolytic-cleavage-of-peroxides-and-oxidation-of-amines-sulfides-and-related-species"><a href="#heterolytic-cleavage-of-peroxides-and-oxidation-of-amines-sulfides-and-related-species">3.3.1 Heterolytic Cleavage of Peroxides and Oxidation of Amines, Sulfides, and Related Species</a></h3>
</td><td>

<h3 id="过氧化物的异裂与胺硫化物极其相关物类的氧化"><a href="#过氧化物的异裂与胺硫化物极其相关物类的氧化">3.3.1 过氧化物的异裂与胺、硫化物极其相关物类的氧化</a></h3>
</td></tr>
<tr><td>

<p>Hydrogen peroxide is a key intermediate formed during the activation of molecular oxygen via the Udenfriend reaction. In addition to its role as the precursor for HO<sup>•</sup> free radical via the Fenton reaction, hydrogen peroxide can also undergo non-radical-mediated oxidation. The most pharmaceutically significant oxidation pathway in this category is the oxidation of amines, sulfides, and similar functional groups containing nucleophilic lone electron pairs. The key step in this oxidation is an SN2 nucleophilic attack by the N and S moieties of the oxidation substrates (Scheme 3.11).</p>
</td><td>

<p>过氧化氢是 Udenfriend 反应中氧分子活化时的关键中间体。除了经历芬顿反应转化为羟基自由基外，过氧化氢还能参与非自由基氧化反应。药物降解中，胺、硫化物和类似的含孤对电子的亲核性基团即可发生此种氧化反应。此反应的关键在于底物的 N 或 S 原子以 S<sub>N</sub>2 机理进攻过氧化氢(Scheme 3.11)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.11.png" alt="Scheme 3.11  " /><p class="caption"><span class="pic-ref">Scheme 3.11</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Hence, the rate of this SN<sub>2</sub> reaction depends upon electronic and steric factors of the oxidation substrates. Among all alkyl amines, tertiary amines would most easily undergo this pathway,<span class="cite-ref"><sup>[62]</sup></span> because the lone pair of electrons on the tertiary amines are the most nucleophilic owing to the presence of three electron-pushing alkyl groups. Toney et al. measured the oxidation rates of two tertiary amines (N-lauryl morpholine and dimethyllaurylamine) and one secondary amine (piperidine) by hydrogen peroxide.<span class="cite-ref"><sup>[63]</sup></span> It was found that N-lauryl morpholine oxidizes the fastest, while piperidine oxidizes the slowest. The results are consistent with the SN<sub>2</sub> reaction mechanism: N-lauryl morpholine has both the electronic and steric advantage in that it is a cyclized tertiary amine which should be less sterically hindered than the acyclic tertiary amine, dimethyllaurylamine. If at least one of the three alkyl groups is replaced by an aryl group, the lone pair electrons of the amino group in the resulting aromatic amines can conjugate with the aryl group and thus become much less nucleophilic. Consequently, aromatic amines usually could not be oxidized by hydrogen peroxide via the nucleophilic route without the use of catalysts.<span class="cite-ref"><sup>[64]</sup></span></p>
</td><td>

<p>此 S<sub>N</sub>2 反应的反应速率取决于底物的电负性和空间位阻。烷基胺中，叔胺的 N 原子上连接有三个推电子的烷基，亲核性最强最容易发生此反应。<span class="cite-ref"><sup>[62]</sup></span> Toney 等人测定了过氧化氢氧化两个叔胺（N-十二烷基吗啉和二甲基十二烷基胺）和一个仲胺（哌啶）的反应速率。<span class="cite-ref"><sup>[63]</sup></span> 研究发现，N-十二烷基吗啉的氧化速度最快，哌啶最慢。此实验结果符合 S<sub>N</sub>2 机理：N-十二烷基吗啉亲核性最强，且 N 原子处于环上，较之二甲基十二烷基胺空间位阻更小。若将三个烷基中的一个换做芳香基团，氮原子的孤对电子将与芳环构成共轭，其亲核性锐减。因此，没有催化剂存在时，芳香胺不足以进行 S<sub>N</sub>2 亲核进攻，故无法被过氧化氢氧化。<span class="cite-ref"><sup>[64]</sup></span></p>
</td></tr>
<tr><td>

<p>In a kinetic study of N-oxidation of 4-substituted N,N-dimethylanilines by hydrogen peroxide in the presence of rhenium trioxide as the catalyst, Zhu and Esperson found that the oxidation is inhibited by electron-withdrawing groups in the 4-position.<span class="cite-ref"><sup>[65]</sup></span> This finding is again consistent with the SN<sub>2</sub> reaction mechanism. For the same reason, pyridine and pyridine-like moieties are not oxidized by hydrogen peroxide alone; for example, imatinib contains a piperizine ring, a pyridine, and a pyrimidine moiety and only the two nitrogens in the piperizine ring are oxidized under excessive stress with 10% hydrogen peroxide.<span class="cite-ref"><sup>[66]</sup></span> When amines are protonated at low pH, their nucleophilicity is greatly reduced, resulting in a much muted reactivity toward autooxidation.<span class="cite-ref"><sup>[67]</sup></span> On the other hand, the ability of non-ionizable thioethers (sulfides) and related compounds, such as disulfides, to undergo nucleophilic oxidation by hydrogen peroxide is not negatively impacted by low pH.</p>
</td><td>

<p>Zhu 和 Esperson 在三氧化铼为催化剂，过氧化氢氧化对位取代的 N,N-二甲基苯胺的反应动力学研究中发现：在对位引入吸电子基团可使氧化反应更容易发生。<span class="cite-ref"><sup>[65]</sup></span> 此结果更有力的支持了 S<sub>N</sub>2 机理。同理，无催化剂时，吡啶或类似结构无法被过氧化氢氧化。例如，imatinib 含有一个哌啶环，一个吡啶环和一个嘧啶环，在 10% 的双氧水强制降解下，只有哌啶的两个氮原子被氧化了。<span class="cite-ref"><sup>[66]</sup></span> 较低 pH 下，胺的 N 原子将结合质子，其亲核性锐减，自然氧化作用亦随之减弱。<span class="cite-ref"><sup>[67]</sup></span> 此外，硫醚极其相关物类，比如，二硫化物，无法质子化，故其与过氧化氢的反应不受低 pH 抑制。</p>
</td></tr>
<tr><td>

<p>Hydrogen peroxide formed during autooxidation may react or interact with certain components of a drug formulation or stress system to yield stronger oxidants. Specifically, hydrogen peroxide can react with carboxylic acids, bicarbonate, and organonitriles (particularly acetonitrile, owing to its widespread use) to yield carboxyl peracids,<span class="cite-ref"><sup>[68]</sup></span> peroxymonocarbonate,<span class="cite-ref"><sup>[69-71]</sup></span> and peroxycarboximidic acids,<span class="cite-ref"><sup>[72]</sup></span> respectively. The resulting species owe their increased oxidation capability to the formation of a better leaving group than hydroxide/water in each case, facilitating the nucleophilic oxidative degradation (Scheme 3.12).</p>
</td><td>

<p>自然氧化过程中生成过氧化氢可能与制剂或强制降解体系中的某些物质反应而产生更强的氧化剂。特别是，过氧化氢能与羧酸、碳酸氢盐、腈（一般为乙腈，其用途最广）反应分别生成过氧酸<span class="cite-ref"><sup>[68]</sup></span>、过一碳酸<span class="cite-ref"><sup>[69-71]</sup></span>、过氧亚氨酸(peroxycarboximidic acid)<span class="cite-ref"><sup>[72]</sup></span>。此三者皆有比氢氧根离子或水更好的离去基，其氧化性更强，使得亲核性氧化降解更容易发生(Scheme 3.12)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.12.png" alt="Scheme 3.12  " /><p class="caption"><span class="pic-ref">Scheme 3.12</span>  </p>
</div>
</td></tr>
<tr><td>

<p>These &quot;activated&quot; forms of hydrogen peroxide have been used in synthetic organic chemistry to generate N-oxides, epoxides, sulfoxides, and sulfones, and so on. This can also explain why different levels of oxidation or oxidative degradation profiles may be obtained, dependent upon the choice of organic solvent (e.g. acetonitrile versus methanol) and other reagents, during forced degradation of a drug substance using hydrogen peroxide.</p>
</td><td>

<p>在有机合成化学中，这些过氧化氢的活化形态早已被用来合成胺氧化物、环氧化物、砜等。这也解释了在使用过氧化氢的强制降解实验中，不同的溶剂（比如乙腈或甲醇）和试剂会引起不同程度的氧化降解。</p>
</td></tr>
<tr><td>

<h3 id="heterolytic-cleavage-of-peroxides-and-formation-of-epoxides"><a href="#heterolytic-cleavage-of-peroxides-and-formation-of-epoxides">3.3.2 Heterolytic Cleavage of Peroxides and Formation of Epoxides</a></h3>
</td><td>

<h3 id="过氧化物的异裂与环氧化物的生成"><a href="#过氧化物的异裂与环氧化物的生成">3.3.2 过氧化物的异裂与环氧化物的生成</a></h3>
</td></tr>
<tr><td>

<p>Although epoxides can be formed via a free radical pathway as discussed in Section 3.2.4, they can also be formed through non-radical pathways.</p>
</td><td>

<p>环氧化物既能依照小节 3.2.4 所述的自由基机理产生，也能经由非自由基参与的途径产生。</p>
</td></tr>
<tr><td>

<p>Hydroperoxides, in particular the activated forms of hydrogen peroxide, e.g. peracids<span class="cite-ref"><sup>[73]</sup></span> and related compounds,<span class="cite-ref"><sup>[74]</sup></span> can react directly with electron-rich double bonds via an electrophilic oxygen transfer process, leading to the formation of epoxides, as shown in Scheme 3.13.</p>
</td><td>

<p>过氧化物，尤其是过氧化氢的活化形态（例如过氧酸<span class="cite-ref"><sup>[73]</sup></span>及其相关物类），可与富电子的双键直接反应：亲核进攻后发生氧转移，生成环氧化物。见 Scheme 3.13。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.13.png" alt="Scheme 3.13  " /><p class="caption"><span class="pic-ref">Scheme 3.13</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The double bonds in olefins that contain electron-withdrawing groups are electron-deficient. in such cases, the epoxidation can proceed through a nucleophilic oxygen transfer process. The epoxidation of α,β-unsaturated car-bonyl compounds with hydroperoxides under basic conditions, that is, the Weitz-Scheffer reaction<span class="cite-ref"><sup>[75]</sup></span> is such an example (Scheme 3.14). According to the mechanism proposed by Bunton and Minkoff,<span class="cite-ref"><sup>[76]</sup></span> this oxidation proceeds via a two-step, addition and ring-closure mechanism shown in Scheme 3.14.</p>
</td><td>

<p>烯烃的双键上若连有吸电子基，则成为贫电子双键。此时，可经历如上的亲核氧转移历程发生环氧化。碱性条件下，以过氧化物实现 α,β-不饱和羰基化合物环氧化的反应，即所谓的 Weitz-Scheffer 反应<span class="cite-ref"><sup>[75]</sup></span>，就是一个实例。依据 Bunton 和 Minkoff 所提出的机理<span class="cite-ref"><sup>[76]</sup></span>，此过程经历两步，加成与关环，详见 Scheme 3.14。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.14.png" alt="Scheme 3.14  " /><p class="caption"><span class="pic-ref">Scheme 3.14</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The epoxide formed may be isolatable but frequently would decompose further. Oxidative degradation involving heterolytic peroxide cleavage will be discussed further in Sections 3.5.2 with representative drugs containing oxidizable carbon-carbon double bonds.</p>
</td><td>

<p>生成的环氧化物经常会进一步分解，但有可能被分离出来。涉及过氧化物异裂的氧化降解将在小节 3.5.2 中结合含有易氧化的碳碳双键的典型药物分子做进一步讨论。</p>
</td></tr>
<tr><td>

<h2 id="carbanionenolate-mediated-autooxidation-base-catalyzed-autooxidation"><a href="#carbanionenolate-mediated-autooxidation-base-catalyzed-autooxidation">3.4 Carbanion/enolate-mediated Autooxidation (Base-catalyzed Autooxidation)</a></h2>
</td><td>

<h2 id="碳负离子烯醇负离子介导的自然氧化碱催化自然氧化"><a href="#碳负离子烯醇负离子介导的自然氧化碱催化自然氧化">3.4 碳负离子/烯醇负离子介导的自然氧化（碱催化自然氧化）</a></h2>
</td></tr>
<tr><td>

<p>In contrast to free radical-mediated autooxidation, carbanion/enolate-mediated autooxidation is much less known for its role in the autooxidation of drugs. Interestingly, oxidation of carbanion/enolate by molecular oxygen (i.e. autooxidation) has been known since 1930s with its main utility being in synthetic organic chemistry.<span class="cite-ref"><sup>[77]</sup></span> More recently, some of the syntheses based on carbanion/enolate autooxidation are promoted as &quot;green chemistry&quot; because the oxidizing agent is molecular oxygen rather than hazardous metal-based oxidizing agents.<span class="cite-ref"><sup>[78]</sup></span> Since the vast majority of the carbanion/enolate species are generated using strong bases, the process is also called &quot;base-catalyzed auto-oxidation&quot;. For drug substances containing somewhat &quot;acidic&quot; carbonated protons (CH<sub>n</sub>, n is typically 1 to 2), the acidic CHn can be slightly deprotonated by a relatively weak base or a general base in the drug formulation, which could result in the formation of impurities exceeding the International Conference on Harmonisation (ICH) thresholds for impurity identification or qualification (typically between 0.1% and 0.5%, dependent upon its daily maximum dose and potency for non-genotoxic impurities). Carbanion/enolate-mediated autooxidation (base-catalyzed autooxidation) can be described by Scheme 3.15.</p>
</td><td>

<p>自由基介导的自然氧化早已广为人知，但碳负离子/烯醇负离子介导的自然氧化在药物降解中的重要意义，却知之者甚少。更有趣的是，自 19 世纪三十年代开始，碳负离子/烯醇负离子介导的自然氧化就广泛应用于有机合成。<span class="cite-ref"><sup>[77]</sup></span> 此种合成方法无需有毒的重金属氧化剂而直接使用氧气，符合“绿色化学”的要求，近年来尤受瞩目。<span class="cite-ref"><sup>[78]</sup></span> 由于想要产生碳负离子/烯醇负离子，往往要在体系中加入强碱，故又名“碱催化自然氧化”。含有酸性氢的药物分子，会因为制剂中存在的弱碱或广义碱的影响而失去质子（即化学家所谓的“拔氢”），进而产生杂质乃至超过 ICH 所规定的鉴定阈值或界定阈值（一般为 0.1~0.5%，依据药物的每日最大剂量与杂质毒性确定）。碳负离子/烯醇负离子介导的自然氧化（碱催化自然氧化）的机理见 Scheme 3.15。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.15.png" alt="Scheme 3.15  " /><p class="caption"><span class="pic-ref">Scheme 3.15</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Once the carbanion/enolate is formed by deprotonatation of an &quot;acidic&quot; carbon-bonded proton, it can quickly react with molecular oxygen to give the organic peroxide. The latter usually further decomposes to yield a number of final degradation products, including alcohol, ketone, anhydride, carboxylic acid, and rearrangement products depending upon the structure of the organic peroxide intermediate and other factors such as pH and solvent. Since a car-banion/enolate is usually a singlet species, its apparent swift reaction with the triplet ground state molecular oxygen appears to contradict the spin conservation rule.<span class="cite-ref"><sup>[4,79-81]</sup></span> To overcome this controversy, it was proposed that the carbanion/enolate would transfer an electron to molecular oxygen to form a pair of a carbon-based free radical and superoxide anion radical in a &quot;caged&quot; complex, which is then followed by spin inversion and subsequent combination to produce the peroxide or peroxide anion. On the other hand, whether or not the caged process exists, the overall rate of the carbanion/enolate-mediated autooxidation appears much faster than a usual free radical-mediated process and apparently displays no characteristics typical of a free radical-mediated reaction.<span class="cite-ref"><sup>[7,82,83]</sup></span></p>
</td><td>

<p>酸性氢容易被碱拔掉，碳负离子/烯醇负离子一旦产生，可与氧气迅速反应生成过氧化物。过氧化物分解则给出诸多降解产物，例如醇、酮、酸酐、羧酸等。若 pH、溶剂等条件适宜，也可能会出现过氧化物的重排反应。碳负离子/烯醇负离子一般是单线态的，但它却能与三线态的氧分子迅速反应，这似乎有悖于自旋守恒规则。<span class="cite-ref"><sup>[4,79-81]</sup></span> 为消除争议，可认为，碳负离子/烯醇负离子先将一个电子传递给氧分子，从而形成碳自由基和超氧阴离子自由基，且二者处于笼状络合物中，随后即遵从自旋守恒发生双自由基终止产生过氧化物或过氧阴离子。另一方面，无论笼状过渡态是否存在，都无法否认一下事实：碳负离子/烯醇负离子介导的自然氧化的反应速率极快，是常规自由基反应无法比拟的，且此未表现出任何自由基反应的代表性特征。<span class="cite-ref"><sup>[7,82,83]</sup></span></p>
</td></tr>
<tr><td>

<h2 id="oxidation-pathways-of-drugs-with-various-structures"><a href="#oxidation-pathways-of-drugs-with-various-structures">3.5 Oxidation Pathways of Drugs with Various Structures</a></h2>
</td><td>

<h2 id="结构各异的药物分子的氧化途径"><a href="#结构各异的药物分子的氧化途径">3.5 结构各异的药物分子的氧化途径</a></h2>
</td></tr>
<tr><td>

<p>The origins and mechanisms of oxidative degradation of drugs of several major types of autooxidation mechanisms have been described in Sections 3.2 to 3.4. in this section, specific oxidation pathways of drugs with various functional groups and functional structures will be discussed in relation to each type of the oxidation mechanism. Note that the same type of functional group may undergo different pathways under different conditions including different dosages. For example, the carbon-carbon double bonds can undergo allylic oxidation as well as epoxidation. On the other hand, formation of the same degradant may derive from multiple degradation pathways. Epoxide, for example, can be formed from both radical and non-radical pathways.</p>
</td><td>

<p>在前面章节中已经讨论了主要的几种自然氧化机理所构成的药物分子的氧化降解途径及其根源。本节中，我们将详细讨论，依照上述机理，不同基团、不同结构的药物分子的具体降解途径。要注意，不同条件（比如剂型不同）下，相同的官能团亦有可能经历不同的氧化途径。例如，碳碳双键既能发生烯丙位氧化也能发生环氧化反应。另一方面，相同的降解产物有可能来自于不同的降解机理，比如在自由基途径和非自由基途径中都可生成环氧化物。</p>
</td></tr>
<tr><td>

<h3 id="allylic--and-benzylic-type-positions-susceptible-to-hydrogen-abstraction-by-free-radicals"><a href="#allylic--and-benzylic-type-positions-susceptible-to-hydrogen-abstraction-by-free-radicals">3.5.1 Allylic- and Benzylic-type Positions Susceptible to Hydrogen Abstraction by Free Radicals</a></h3>
</td><td>

<h3 id="烯丙位苄位容易被自由基夺氢"><a href="#烯丙位苄位容易被自由基夺氢">3.5.1 烯丙位、苄位容易被自由基夺氢</a></h3>
</td></tr>
<tr><td>

<p>Drugs containing allylic- and benzylic-type moieties are susceptible to free radical attack, because the resulting carbon-centered radicals are stabilized by conjugation with the nearby double bond or aromatic ring. The carbon-centered radicals usually react with O<sub>2</sub> at an extremely fast rate (approaching the diffusion controlled rate). Nonetheless, carbon-centered radicals that are stabilized by extensive resonance, such as triphenylmethyl and 9-phenyl-fluorenyl, have greatly reduced reactivity with O<sub>2</sub>. Other factors can also have an impact on the reactivity with O<sub>2</sub>. For example, Bejan et al. reported that the benzylic radical derived from 2-coumaranone, which has a lactone functionality next to the radical, completely lacks any reactivity with O<sub>2</sub> (Figure 3.2).<span class="cite-ref"><sup>[84]</sup></span></p>
</td><td>

<p>药物分子中的烯丙位或苄位容易受自由基进攻，生成的碳自由基受到双键或芳环的共轭作用而稳定性更好。碳自由基能非常快地与 O<sub>2</sub> 反应（接近扩散控制速率——即反应速率极快，以至于限制反应速率的最主要因素是反应物在反应介质中的转移速度）。此外，大范围共振（比如，三苯甲基、9-苯基芴基）能使碳自由基更稳定，但也能强烈地降低与 O<sub>2</sub> 的反应活性。例如，Bejan 等人报道了由 2-coumaranone 衍生出的苄位自由基(图 3.2)，此自由基恰邻近内酯键，几乎完全不与 O<sub>2</sub> 反应。<span class="cite-ref"><sup>[84]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.2.png" alt="Figure 3.2   2-coumaranone 衍生物所对应的苄位自由基 Structure of the benzylic radicals derived from 2-coumaranone derivatives." /><p class="caption"><span class="pic-ref">Figure 3.2</span>   2-coumaranone 衍生物所对应的苄位自由基<br /> Structure of the benzylic radicals derived from 2-coumaranone derivatives.</p>
</div>
</td></tr>
<tr><td>

<p>During pharmaceutical development of a novel drug candidate, <strong>TCH346</strong>, for neurodegenerative disorders, a degradant devoid of the original amine moiety was found during long term and accelerated stability studies of a tablet formulation.<span class="cite-ref"><sup>[85]</sup></span> The authors proposed the degradation mechanism shown in Scheme 3.16.</p>
</td><td>

<p>用于治疗神经退行性紊乱的备选药物 <strong>TCH346</strong> 的开发过程中，片剂的长期与加速稳定性研究发现了丧失原有胺结构的降解产物。<span class="cite-ref"><sup>[85]</sup></span> 论文作者认为其降解机理如下 Scheme 3.16。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.16.png" alt="Scheme 3.16   Reproduction from Reference 85 with permission." /><p class="caption"><span class="pic-ref">Scheme 3.16</span>   Reproduction from Reference 85 with permission.</p>
</div>
</td></tr>
<tr><td>

<p>In this drug substance, the allylic position is quite susceptible to H abstraction by a free radical generated in autooxidation, since the radical formed can be stabilized by an extended conjugated system. The allylic radical should readily react with O<sub>2</sub> to give a peroxide which would in turn produce the alcohol intermediate. It is possible that the peroxide could directly decompose to give the final aldehyde degradant, while producing a hydroxylamine as the leaving group. Hence, an alternate degradation mechanism (Scheme 3.17, pathway a) can be proposed.</p>
</td><td>

<p>由于共轭能使产物更稳定，此药物结构中的烯丙位很容易受自由基夺氢而发生自然氧化。烯丙基自由基随即与 O<sub>2</sub> 反应形成过氧化物，羟基胺作为离去基团，最终产生醛。故此可提出 Scheme 3.17 途径a 所示的可能降解机理。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.17.png" alt="Scheme 3.17  " /><p class="caption"><span class="pic-ref">Scheme 3.17</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In this particular case, the benzylic position is also α to the tertiary amine moiety. This type of structural moiety can also form an aminium radical cation intermediate through a one electron transfer oxidation of the nitrogen atom, which will give the same aldehyde degradant while producing an amine as the leaving group (Scheme 3.17, pathway b). The degradant distribution in the latter case is the same as that proposed by the original researchers. Since the structure of the leaving group was not determined in the original study, it is not possible to tell which degradation pathway is more likely. There will be a more detailed discussion of the autooxidation mechanism via the aminium radical cation intermediate in Section 3.5.3.3.</p>
</td><td>

<p>此例中，苄位的同时也是叔胺的 α 位（即此亚甲基连接了芳环与叔胺）。这种结构中的 N 原子可发生单电子转移而被氧化为胺自由基正离子，这将产生仲胺作为离去基，最终氧化产物同样为醛(Scheme 3.17, 途径b)。原论文作者同意提出了这一降解途径，但并未测定离去基团的结构，故无法分辨这两途径那种更合理。胺自由基正离子介导的自然氧化降解将在小节 3.5.3.3 详细讨论。</p>
</td></tr>
<tr><td>

<p>Clopidogrel bisulfate, the active pharmaceutical ingredient (API) of the second best selling drug Plavix, contains a moiety that is similar to that in <strong>TCH346</strong> above: a benzylic-type 3-thiothenylmethyl position that is also α to a tertiary amine functionality. Recently, a significant new oxidative degradant was observed in the clopidogrel bisulfate drug substance and drug product.<span class="cite-ref"><sup>[86]</sup></span> This new degradant elutes close to the void volume in the clopidogrel bisulfate USP method<span class="cite-ref"><sup>[87]</sup></span> due to the polar iminium cation moiety. Despite the thorough structure characterization carried out by the original workers, no formation mechanism was proposed for this new degradant. Based on the similarity of the key functionalities between clopidogrel bisulfate and that of <strong>TCH346</strong>, formation of the new oxidative degradant can proceed through either of the two radical pathways (Scheme 3.18) via intermediates that are analogous to those in Scheme 3.17.</p>
</td><td>

<p>第二大畅销药物 Plavix 的活性药物成份(API)，Clopidogrel bisulfate 含有类似于 <strong>TCH346</strong> 的结构：苄位同时是叔胺的 α 位。不久前，在原料药和制剂都捕捉到了一个新的氧化降解产物。<span class="cite-ref"><sup>[86]</sup></span> 其结构中的亚胺离子使得此分子极性较大，以至于在 USP 检测方法下，此降解产物的保留时间接近死时间<span class="cite-ref"><sup>[87]</sup></span> 作者表征了其结构却没有提出相应的生成机理。考虑到 Clopidogrel bisulfate 与 <strong>TCH346</strong> 的结构相似，我们可推测其降解机理类似，可能存在两种自由基途径(Scheme 3.18)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.18.png" alt="Scheme 3.18  " /><p class="caption"><span class="pic-ref">Scheme 3.18</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The molecule of morphine contains both a benzylic (C10) and an allylic (C14) position. Two degradants resulting from C10 oxidation were observed in morphine sulfate drug substance as well as in several pharmaceutical preparations.<span class="cite-ref"><sup>[88-90]</sup></span> The formation of these two degradants, 10α-Hydroxy and 10-oxomorphine, is probably via the pathway shown in Scheme 3.19.</p>
</td><td>

<p>Morphine 分子中也存在苄位(C10)和烯丙位(C14)。在 morphine sulfate 原料药及其多种药物制品中皆检测到了 C10 位受氧化的降解产物。<span class="cite-ref"><sup>[88-90]</sup></span> 降解产物 10α-hydroxymorphine 和 10-oxomorphine 的可能生成机理见 Scheme 3.19。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.19.png" alt="Scheme 3.19  " /><p class="caption"><span class="pic-ref">Scheme 3.19</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The stability results showed that 10-oxomorphine continuously increased over time, while 10α-Hydroxymorphine remained relatively unchanged. This phenomenon is consistent with the above mechanism where 10α-hydroxymorphine is an intermediary degradant leading to the terminal degradant, 10-oxomorphine. Under chemical transformation conditions, no oxidation was seen on C14 position,<span class="cite-ref"><sup>[90]</sup></span> indicating the C14 allylic position is less reactive than the benzylic C10 position towards what is presumed to be radical-mediated autooxidation. On the other hand, the phenolic moiety of morphine (and related drugs, e.g., naloxone, nalbuphine, and oxymorphone) can undergo oxidative 2,2'-dimerization in solutions to yield primarily 2,2'-morphine dimer (pseudomorphine). Refer to Section 3.5.9 for a more detailed discussion on the oxidation mechanism of drugs containing a phenolic moiety.</p>
</td><td>

<p>稳定性试验显示，10-oxomorphine 随时间不断增加；10α-hydroxymorphine 则基本不变。这一现象说明，10α-hydroxymorphine 乃是氧化过程的中间产物，它将进一步被氧化为终产物 10-oxomorphine。在足以引起化学变质的条件下依然未观察到 C14 位氧化产物<span class="cite-ref"><sup>[90]</sup></span>，这足以说明烯丙位(C14)远不如苄位(C10)活泼。另一方面，在溶液中，morphine （及其相关药物，如 naloxone、nalbuphine、oxymorhine）的酚环可发生氧化性二聚生成 2,2'-morphine dimer (pseudomorphine)。小节 3.5.9 中将详述含酚环药物的此种氧化机理。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.3.png" alt="Figure 3.3   Ezlopitant 的结构式，箭头指示了过氧化反应位点。 The structure of ezlopitant. The arrow indicates where peroxidation occurs." /><p class="caption"><span class="pic-ref">Figure 3.3</span>   Ezlopitant 的结构式，箭头指示了过氧化反应位点。<br /> The structure of ezlopitant. The arrow indicates where peroxidation occurs.</p>
</div>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.20.png" alt="Scheme 3.20  " /><p class="caption"><span class="pic-ref">Scheme 3.20</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Other examples of autooxidation involving a diene functionality include lovastatin and simvastatin, which are the first and second generation HMG-CoA reductase inhibitors used for the treatment of hypercholesterolemia. Both drug substances have a diene functionality embedded in their ten-membered fused ring core structures. This diene functionality is particularly reactive towards free radical-mediated autooxidation in both solid and solution states.<span class="cite-ref"><sup>[94,95]</sup></span> Owing to the presence of various resonance forms of the initial radical generated and their reactions with molecular oxygen and/or among themselves, a great number of oxidative degradants can be produced (Scheme 3.21).</p>
</td><td>

<p>可举出其他包含二烯结构的药物分子自然氧化的例子，比如用于治疗高胆固醇血症的羟甲基戊二酸单酰辅酶A还原酶抑制剂(HMG-CoA reductase inhibitor)，第一代的 lovastatin、第二代的 simvastatin，其十元稠环母核中皆有共轭二烯结构。无论在固态还是溶液态，此二烯结构都可发生自由基氧化。<span class="cite-ref"><sup>[94,95]</sup></span> 所产生的自由基将呈现复杂的共振式，极性结构进而与 O<sub>2</sub> 或彼此间反应生成一系列复杂的降解产物(Scheme 3.21)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.21.png" alt="Scheme 3.21  " /><p class="caption"><span class="pic-ref">Scheme 3.21</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Structural variations of the benzylic and allylic moieties, such as the CH positions that are α to a carbon-heteroatom double bond or a to a heterocyclic aromatic ring, are also susceptible to the same free radical-mediated auto-oxidation. For example, the anti-psychotic drug risperidone (Figure 3.4) contains a fused pyrimidin-4-one ring (ring A). The α-position (9-position) next to the heterocyclic pyrimidin-4-one ring was found to undergo autooxidation in bulk drug as well as in a tablet formulation, resulting in the formation of 9-hydroxyrisperidone as the main degradant.<span class="cite-ref"><sup>[96]</sup></span> This degradant is also a metabolite of the drug.<span class="cite-ref"><sup>[97]</sup></span> On the other hand, N-oxidation on the middle piperidine ring also occurred; the N-oxide was the second most significant degradant.</p>
</td><td>

<p>苄位或烯丙位的各种变体，比如碳-杂原子双键的 α 位亚甲基、杂芳环上的亚甲基，同样能发生类似的自由基介导自然氧化反应。比如，抗精神病药物 risperidone (图 3.4)，包含一个稠和的尿嘧啶环(ring A)。在原料药和片剂中，此尿嘧啶环的 α 位(C9位)发生自然氧化，生成 9-hydroxyrisperidone <span class="cite-ref"><sup>[96]</sup></span>，这同时也是此药的体内代谢物<span class="cite-ref"><sup>[97]</sup></span>。此外，哌嗪环上的 N 原子可形成胺氧化物，但只是一个次要降解产物。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.4.png" alt="Figure 3.4   risperidone 的结构式。 Structure of risperidone." /><p class="caption"><span class="pic-ref">Figure 3.4</span>   risperidone 的结构式。<br /> Structure of risperidone.</p>
</div>
</td></tr>
<tr><td>

<h3 id="double-bonds-susceptible-to-addition-by-hydroperoxides"><a href="#double-bonds-susceptible-to-addition-by-hydroperoxides">3.5.2 Double Bonds Susceptible to Addition by Hydroperoxides</a></h3>
</td><td>

<h3 id="过氧化氢可加成双键"><a href="#过氧化氢可加成双键">3.5.2 过氧化氢可加成双键</a></h3>
</td></tr>
<tr><td>

<p>During stress testing under autoclaving conditions ( ~115 °C for up to 6 hours), the aqueous solutions of two tricyclic drugs, flupenthixol (dihydrochloride salt) and amitriptyline (hydrochloride salt) in neutral buffers displayed similar degradation patterns.<span class="cite-ref"><sup>[98,99]</sup></span> Analogous tricyclic ketones, namely trifluoromethylthioxanthone and dibenzosuberone, were formed respectively (Scheme 3.22). It would be intuitive to postulate that the formation of the tricyclic ketones may be mediated through an epoxide intermediate of the parent drugs via the electrophilic oxygen transfer process as shown in the upper pathway of Scheme 3.22, the mechanism of which has been discussed above in Section 3.3.2. Nevertheless, such an intermediate could not be isolated in either case, although an epoxide formed from an intermediary degradant of flupenthixol was isolated. The latter epoxide quickly decomposed to the corresponding tricyclic ketone, trifluoromethylthioxanthone upon exposure to air. This observation, along with the fact that a few other intermediary degradants were also seen during the stress test, led the authors to propose a stepwise degradation pathway leading to the final formation of the tricyclic ketones (Scheme 3.22).</p>
</td><td>

<p>高压灭菌条件（~115°C，保持 6 小时）的强制降解研究发现，两个三环药物分子：flupenthixol (二盐酸盐)、amitriptyline (盐酸盐)，在中性缓冲溶液中的降解行为类似。<span class="cite-ref"><sup>[98,99]</sup></span> 分别降解产生 trifluoromethylthioxanthone 和 dibenzosuberone，此二者结构接近(Scheme 3.22)。回忆一下小节 3.3.2 中讨论的亲电氧转移机理，凭直觉分析，会很自然地认为这两个分子的降解是经历了环氧化物中间体。但是，只分离出了 flupenthixol 的环氧化物中间体；amitriptyline 所对应的环氧化物在空气中即迅速分解生成相应的酮。与此同时，在强制降解中还观测到了其他一些中间降解产物。据此作者提出了 Scheme 3.22 所示的分步降解途径。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.22.png" alt="Scheme 3.22  " /><p class="caption"><span class="pic-ref">Scheme 3.22</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the course of a liquid and tablet formulation study of tiagabine, a potent inhibitor of gamma-aminobutyric acid (GABA) uptake for the treatment of epilepsy, two major degradants, dihydroxytiagabine and ketotiagabine, were observed.<span class="cite-ref"><sup>[100]</sup></span> The formation of dihydroxytiagabine is most likely to take place via a transient epoxide intermediate, while ketotiagabine appears to be a further dehydration degradant of dihydroxytiagabine (Scheme 3.23).</p>
</td><td>

<p>Tiagabine 是强效的γ-氨基丁酸(gamma-aminobutyric acid, GABA)摄取抑制剂，用于治疗癫痫症。在其片剂和液体制剂研究中，观测到两个主要降解产物：dihydroxytiagabine 和 ketotiagabine。<span class="cite-ref"><sup>[100]</sup></span> Dihydroxytiagabine 很有可能是由环氧化物水解而来，而 ketotiagabine 则是 dihydroxytiagabine 的脱水产物——Pinacol 重排(Scheme 3.23)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.23.png" alt="Scheme 3.23  " /><p class="caption"><span class="pic-ref">Scheme 3.23</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The indole ring is an important functional moiety that is present in the amino acid, tryptophan; its UV absorption property is mostly responsible for the absorbance of proteins at 280 nm, a wavelength widely used for protein detection and assays. The indole ring also exists in many natural products, fragrances, as well as drugs. Part of the fused pyrrole ring in indole can be considered to be an embedded enamine moiety. As such, the double bond in the fused pyrrole ring is quite electron-rich and hence susceptible to oxidation by hydroperoxides. The oxidation proceeds via nucleophilic attack of the double bond with hydroperoxide (or electrophilic oxygen transfer from the perspective of hydroperoxide). The resulting epoxide usually further degrades into various final degradants depending upon the structures connecting to the indole ring. For example, epoxides of simple alkyl-substituted indoles decompose to 2-oxindoles<span class="cite-ref"><sup>[101]</sup></span> (Scheme 3.24).</p>
</td><td>

<p>吲哚环是重要的官能团，广泛出现于天然产物、香料和药物。色氨酸即包含吲哚环结构，其紫外最大吸收波长为 280 nm，恰是多肽检测和定量的最通用波长。而吲哚环的 N 原子可看做是嵌于环内的烯胺，此富电子烯键易受过氧化物氧化。过氧化物亲核进攻碳碳双键，或发生亲电氧转移，从而生成环氧化产物。此环氧化物往往会进一步降解生成相应的最终降解产物。例如，简单烷基取代的吲哚会环氧化然后进一步分解生成 2-氧代吲哚(Scheme 3.24)。<span class="cite-ref"><sup>[101]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.24.png" alt="Scheme 3.24  " /><p class="caption"><span class="pic-ref">Scheme 3.24</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Nevertheless, during a hydrogen peroxide stress study of indomethacin, a non-steroidal anti-inflammatory drug containing a 5-methoxyindole ring, two major degradants were produced, which can be rationalized as being formed from the epoxide intermediate through pathways that differ from Scheme 3.24.<span class="cite-ref"><sup>[102]</sup></span> The expected methyl 2,3-shift (pathway a, Scheme 3.25) did not occur.</p>
</td><td>

<p>此外，非甾体抗炎药 indomethacin 结构中含有以 5-甲基吲哚环，在其双氧水强制降解研究中发现了两个主要降解产物。其生成机理的合理的解释是同样经历环氧中间体，但下一步的反应不同于 Scheme 3.24。<span class="cite-ref"><sup>[102]</sup></span> 出乎意料的是，甲基未发生 2,3-迁移(Scheme 3.25，途径 a)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.25.png" alt="Scheme 3.25  " /><p class="caption"><span class="pic-ref">Scheme 3.25</span>  </p>
</div>
</td></tr>
<tr><td>

<p>As mentioned above, drugs containing electron-deficient double bonds can undergo epoxidation through nucleophilic oxygen transfer from hydroperoxides. One example can probably be found in the hydrogen peroxide stress of tetrazepam, a benzodiazepine used clinically as a myorelaxant. When the stress was conducted at 40 °C in dark, the epoxide was formed as the only degradant.<span class="cite-ref"><sup>[103]</sup></span> Owing to the presence of the conjugated imine moiety, the nucleophilic oxygen transfer in Scheme 3.26 can be proposed.</p>
</td><td>

<p>前文已述，富电子双键受过氧化物亲核，发生氧转移而生成环氧化物。另一个例子是，苯二氮卓类肌肉松弛剂 tetrazepam 的双氧水强制降解实验：40°C，避光，则降解产物只有环氧化产物。<span class="cite-ref"><sup>[103]</sup></span> 考虑到共轭亚胺的存在，可提出 Scheme 3.26 所示的亲核氧取代机理。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.26.png" alt="Scheme 3.26  " /><p class="caption"><span class="pic-ref">Scheme 3.26</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the degradation of a tablet formulation of tetrazepam, however, the epoxide was observed only as a minor degradant, while the main degradation that occurred was oxidation at the 3'-allylic position. This suggests that the degradation pathway to the epoxide degradant in a tablet formulation may proceed through a radical-mediated process. Indeed, stress on a tetrazepam solution with a radical initiator, azobisisobutyronitrile (AIBN), generated a degradation profile that is quite similar to that of the tablet formulation in an accelerated stability study.<span class="cite-ref"><sup>[103]</sup></span></p>
</td><td>

<p>但在 tetrazepam 的片剂中，环氧化物仅仅是一个次要降解产物。此时的主要降解途径是 3'-烯丙位的氧化。这表明，片剂中的环氧化物可能是来自于自由基氧化过程。事实上，以偶氮二异丁腈(AIBN)为自由基引发剂，tetrazepam 溶液的强制降解情况与其片剂的加速稳定性结果非常相似。<span class="cite-ref"><sup>[103]</sup></span></p>
</td></tr>
<tr><td>

<h3 id="tertiary-amines"><a href="#tertiary-amines">3.5.3 Tertiary Amines</a></h3>
</td><td>

<h3 id="叔胺"><a href="#叔胺">3.5.3 叔胺</a></h3>
</td></tr>
<tr><td>

<h4 id="formation-of-n-oxides-via-nucleophilic-attack-on-hydrogen-peroxide"><a href="#formation-of-n-oxides-via-nucleophilic-attack-on-hydrogen-peroxide">3.5.3.1 Formation of N-oxides via Nucleophilic Attack on Hydrogen Peroxide</a></h4>
</td><td>

<h4 id="亲核进攻过氧化氢生成-n-氧化物"><a href="#亲核进攻过氧化氢生成-n-氧化物">3.5.3.1 亲核进攻过氧化氢生成 N-氧化物</a></h4>
</td></tr>
<tr><td>

<p>In the case of amine oxidation by hydrogen peroxide (or hydroperoxides in general) via a nucleophilic pathway (Scheme 3.27), tertiary alkyl amines are most prone to producing N-oxides by oxidation, as discussed above in Section 3.3.1. The N-oxide of a tertiary alkyl amine is reasonably stable and in most cases, can be isolated.</p>
</td><td>

<p>胺亲核进攻过氧化氢（或其他过氧化物），可被氧化成 N-氧化物(Scheme 3.27)。前文已经提到（小节 3.3.1），叔胺最容易发生此种反应，大多数情况下，烷基叔胺的 N-氧化物很稳定，可被纯化分离。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.27.png" alt="Scheme 3.27  " /><p class="caption"><span class="pic-ref">Scheme 3.27</span>  </p>
</div>
</td></tr>
<tr><td>

<p>A great number of drugs have alkyl tertiary amine functionality and hence are susceptible to autooxidation via the nucleophilic pathway shown in Scheme 3.27 and subsequent degradation pathways. Tertiary amine drugs are also susceptible to free radical-mediated autooxidation via the aminium cation intermediate, which will be discussed in Section 3.5.3.3. A good class example of the nucleophilic oxidative degradation can be found in phenothiazine-derived drugs; there are more than two dozen such drugs according to a search on the web site, http://drugbank.wishartlab.com.<span class="cite-ref"><sup>[104]</sup></span> These drugs all contain a tricylic phenothiazine ring with different substituents on the N atom of the ring. The majority of the N-substituents contain either N,N-disubstituted piperazine ring or acyclic tertiary amine moieties, most of which can be illustrated in Figure 3.5 where the sites for N-oxide formation are indicated by arrows.</p>
</td><td>

<p>大多数含有烷基叔胺的药物分子都容易发生 Scheme 3.27 所示的亲核机理自然氧化反应，进而发生降解。叔胺还容易形成胺正离子自由基，从而发生自由基介导的自然氧化反应，这在将小节 3.5.3.3 中详细讨论。吩噻嗪类药物即是仲胺受亲核进攻发生氧化降解的实例。在网站 http://drugbank.wishartlab.com 上可检索到多达二十余种吩噻嗪类抗精神病药物。<span class="cite-ref"><sup>[104]</sup></span> 此类药物分子结构中皆包含一个不同 N-取代的吩噻嗪环，取代基多为 N,N-二取代的哌嗪环或长链叔胺，参见 图 3.5，箭头指示了 N-氧化物的生成位点。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.5.png" alt="Figure 3.5   吩噻嗪类药物的结构。 Structures of phenothiazine-derived drugs." /><p class="caption"><span class="pic-ref">Figure 3.5</span>   吩噻嗪类药物的结构。<br /> Structures of phenothiazine-derived drugs.</p>
</div>
</td></tr>
<tr><td>

<p>In this class of drugs, the sulfur in the tricyclic ring can compete with the side chain tertiary amines for oxidation during the early stage of autooxidation. Recently, Wang et al. reported a stress study of perphenazine solution in methanol with hydrogen peroxide, which showed the formation of all the three mono-oxidized degradants, that is, perphenazine 17N-oxide, perphenazine 14N-oxide, and perphenazine sulfoxide (Figure 3.6), in addition to lower levels of dioxidized degradants.<span class="cite-ref"><sup>[105]</sup></span></p>
</td><td>

<p>此类药物的吩噻嗪环中的硫原子也可被氧化，在自然氧化的早期，这足以和仲胺侧链的氧化反应形成竞争。最近，Wang 等人报道了甲醇为溶剂，以过氧化氢强制降解 perphenazine 的研究：生成了三种单氧化产物——perphenazine 17N-oxide、perphenazine 14N-oxide、perphenazine sulfoxide (Figure 3.6)，但仅生成少量双氧化产物。<span class="cite-ref"><sup>[105]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.6.png" alt="Figure 3.6   Perphenazine 单氧化降解产物的结构。 Structures of three mono-oxidized degradants of perphenazine." /><p class="caption"><span class="pic-ref">Figure 3.6</span>   Perphenazine 单氧化降解产物的结构。<br /> Structures of three mono-oxidized degradants of perphenazine.</p>
</div>
</td></tr>
<tr><td>

<p>Among these degradants, 17N-oxide is most abundant in the early stage of the stress; interestingly, 17N-oxide is also the most abundant oxidative degradant in a solid formulation containing perphenazine. No oxidation occurred on the aromatic nitrogen, as expected. This result is not in a complete agreement with the stress study of perphenazine reported by Li et al.<span class="cite-ref"><sup>[106]</sup></span> According to this study, perphenazine sulfoxide was observed as the only mono-oxidized degradant.<span class="cite-ref"><sup>[106]</sup></span> The discrepancy may be caused by one of the following two factors: (1) the stress solution used by Li et al. is mostly aqueous, which is quite different from the mostly methanolic solution employed by Wang et al. (2) The two N-oxides might be formed in the stress by Li et al. but not separated by the method they used.<span class="cite-ref"><sup>[106]</sup></span></p>
</td><td>

<p>这些降解产物中，17N-oxide 在强制降解实验的早期生成最多；更有趣的是，它也是固体制剂中最重要的降解产物。和预期的一样，未发现芳香胺的氧化产物。此结果与 Li 等人报道的 perphenazine 强制降解研究略有出入。Li 等人观测到的唯一单氧化降解产物是 perphenazine sulfoxide。<span class="cite-ref"><sup>[106]</sup></span> 而个中缘由可能为：(1) Li 等人进行强制降解时使用的是水溶液，而 Wang 等人使用的却是甲醇溶液。(2) 另外两种单氧化产物在 Li 等人的实验中形成了，却没能被分辨出来。<span class="cite-ref"><sup>[106]</sup></span></p>
</td></tr>
<tr><td>

<p>In a formulation study assessing pH effect on the control of N-oxidative degradants, Freed et al. found that oxidation of alkyl tertiary amines by hydroperoxides (including hydrogen peroxide in a few stress studies) can be inhibited by lowering the pH of stress solutions or by acidifying solid dosage formulations with citric acid.<span class="cite-ref"><sup>[67]</sup></span> In one of their solution stress studies, it is apparent that the pH of the solution needs to be controlled well below the pK<sub>a</sub> of the tertiary amines in order to suppress the N-oxidation effectively.</p>
</td><td>

<p>Freed 等人制在剂研究中考察了 pH 对 N-氧化降解的影响，他们发现降低溶液 pH，或在固体制剂中加入柠檬酸，可抑制过氧化物（包括一些强制降解实验中涉及的过氧化氢）对烷基叔胺的氧化。<span class="cite-ref"><sup>[67]</sup></span> 其中一次溶液态强制降解实验中，似乎需要小心控制溶液 pH 使之低于叔胺的 pK<sub>a</sub> 方才能有效抑制胺氧化反应。</p>
</td></tr>
<tr><td>

<p>For example, the two tertiary amines used in the study, compound <strong>A</strong> (a proprietary drug candidate with partial structure revealed) and raloxifene, displayed significantly different rates of N-oxidation at pH 6: Compound <strong>A</strong> with a pK<sub>a</sub> of 6.45 oxidizes approximately twice as fast as raloxifene which has a calculated pK<sub>a</sub> of 8.67 (Figure 3.7).</p>
</td><td>

<p>另一项研究中，测定了化合物 <strong>A</strong> （部分结构受专利保护的备选药物）与 raloxifene (Figure 3.7) 在 pH=6 时，N-氧化反应速度，两者的 pK<sub>a</sub> 分别为 6.45 和 8.67(估算值)，其结果差异显著：化合物 A 的氧化速度约为 raloxifene 的两倍。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.7.png" alt="Figure 3.7   化合物 A 与 raloxifene。 Compound A and raloxifene." /><p class="caption"><span class="pic-ref">Figure 3.7</span>   化合物 <strong>A</strong> 与 raloxifene。<br /> Compound <strong>A</strong> and raloxifene.</p>
</div>
</td></tr>
<tr><td>

<h4 id="decomposition-of-n-oxides-secondary-and-tertiary-degradation-pathways"><a href="#decomposition-of-n-oxides-secondary-and-tertiary-degradation-pathways">3.5.3.2 Decomposition of N-oxides: Secondary and Tertiary Degradation Pathways</a></h4>
</td><td>

<h4 id="n-氧化物分解伯胺与仲胺的降解途径"><a href="#n-氧化物分解伯胺与仲胺的降解途径">3.5.3.2 N-氧化物分解：伯胺与仲胺的降解途径</a></h4>
</td></tr>
<tr><td>

<p>As discussed previously, alkyl tertiary amine oxides are reasonably stable and can be isolated in most cases. Therefore, further degradation of N-oxides is usually not significant for drug substances and products stored under ICH long term and accelerated stability conditions. Nevertheless, under somewhat excessive reaction conditions such as various stress conditions, N-oxides are capable of degrading further into an array of secondary and tertiary degradants (Scheme 3.28). These further degradation pathways include dealkylation through the iminium ion intermediate, if at least one alkyl group contains an α-H.</p>
</td><td>

<p>如前所述，多数烷基叔胺的 N-氧化物能稳定存在，可分离得到。因此，在 ICH 所规定的长期与加速稳定性条件下，难以明显观测到 N-氧化物的更进一步降解。但在一些特例中，比如某些强制降解条件下，N-氧化物有可能会发生进一步降解(Scheme 3.28)。若氮原子所连烷基带有 α-H，N-氧化物可脱去烷基生成亚胺离子。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.28.png" alt="Scheme 3.28  " /><p class="caption"><span class="pic-ref">Scheme 3.28</span>  </p>
</div>
</td></tr>
<tr><td>

<p>This pathway, in particular demethylation and de-ethylation (R = methyl or ethyl), is significant in the metabolism of alkyl tertiary amine drugs, which is catalyzed by metabolic enzymes,<span class="cite-ref"><sup>[107,108]</sup></span> but is generally insignificant for drugs (particularly in solid dosage forms) under commercial storage conditions which typically follow the ICH long term stability storage conditions. During the course of writing this book, despite repeated searches of the literature, the author has not yet been able to find a meaningful dealkylation case as a result of the nucleophilic oxidation of tertiary amines except for those under somewhat excessive stress conditions.<span class="cite-ref"><sup>[109,110]</sup></span> On the other hand, dealkylation of tertiary amines is possible under free radical-mediated autooxidation conditions, which will be discussed in the next section. Nevertheless, the alkyl groups that are cleaved from the tertiary amines under the latter conditions are usually more complicated than straightforward alkyl groups like methyl and ethyl groups.<span class="cite-ref"><sup>[85,111]</sup></span></p>
</td><td>

<p>在体内代谢中，烷基叔胺受酶催化发生脱烷基化（R = 甲基或乙基）是重要的代谢途径<span class="cite-ref"><sup>[107,108]</sup></span>，但合理贮存（一般会遵从 ICH 所述长期稳定性的保存条件）的药物（特别是固体制剂中）中很难发现此种降解机理。本书写作过程中，剔除重复文献所载实验，笔者未能发现一例有价值的叔胺经亲核氧化生成 N-氧化物后发生脱烷基反应的实例。当然在某些极端强制降解实验确有发现。<span class="cite-ref"><sup>[109,110]</sup></span> 另一方面，叔胺脱烷基化在自由基条件下也可能发生，这将在下一节讨论。但自由基条件下的脱烷基化反应往往会很复杂，远非甲基或乙基时那么直截了当。<span class="cite-ref"><sup>[85,111]</sup></span></p>
</td></tr>
<tr><td>

<p>Other degradation pathways of N-oxides include several thermolytic reactions such as deoxygenation, in which N-oxides are reduced back to the original tertiary amines,<span class="cite-ref"><sup>[112,113]</sup></span> Cope elimination, where an olefin and a hydroxylamine are produced if the N-oxides contain a β-hydrogen,<span class="cite-ref"><sup>[114,115]</sup></span> and Meisenheimer rearrangement.<span class="cite-ref"><sup>[116]</sup></span> These reactions occur only at very high temperature and their only relevance to drug degradation study may be limited to two areas. The examples in the first area include special studies such as the autoclaving studies, discussed in Section 3.5.2, where decomposition of flupenthixol and amitriptyline into the corresponding diene intermediates is very likely via Cope elimination of the two N-oxides as illustrated in Scheme 3.29.</p>
</td><td>

<p>N-氧化物的降解途径还包括热解反应（比如脱氧反应(deoxygenation)，此时 N-氧化物被还原为叔胺）<span class="cite-ref"><sup>[112,113]</sup></span>、Cope 消除（有 β-H 的 N-氧化物发生消除反应生成烯烃和羟胺）<span class="cite-ref"><sup>[114,115]</sup></span>、Meisenheimer 重排<span class="cite-ref"><sup>[116]</sup></span>。这些反应一般需要较高的温度才能进行，故此这些反应只与药物降解研究的两个方面有关。其中一个方面包括诸如高压蒸汽灭菌（详见小节 3.5.2）等特殊情况。例如，蒸汽灭菌条件下，flupenthixol 和 amitriptyline 会降解产生相应的二烯(Scheme 3.29)，这看起来像是经历了 N-氧化物的 Cope 消除。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.29.png" alt="Scheme 3.29  " /><p class="caption"><span class="pic-ref">Scheme 3.29</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The examples in the second area are the thermal degradation pathways that occur during gas chromatography (GC) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) analyses in which processes the temperatures inside the MS detectors can be as high as several hundred degrees centigrade. An example is a recent liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) study of perphenazine 14N-oxide, in which the analyte undergoes all these degradation pathways leading to the formation of various species in the gas phase (Scheme 3.30).<span class="cite-ref"><sup>[105]</sup></span></p>
</td><td>

<p>另一方面则是，药物分子在气相色谱(GC)和大气压化学电离源质谱(APCI-MS)中的热降解行为，此时的质谱检测器中温度高达数百摄氏度。例如，使用高效液相色谱-质谱联用(LC-MS/MS)研究 perphenazine 14N-oxide 时发现，N-氧化物在气态发生了复杂的降解反应生成一系列次级碎片(Scheme 3.30)。<span class="cite-ref"><sup>[105]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.30.png" alt="Scheme 3.30  " /><p class="caption"><span class="pic-ref">Scheme 3.30</span>  </p>
</div>
</td></tr>
<tr><td>

<h4 id="free-radical-mediated-autooxidation-of-tertiary-amines-via-the-aminium-radical-cation-intermediate"><a href="#free-radical-mediated-autooxidation-of-tertiary-amines-via-the-aminium-radical-cation-intermediate">3.5.3.3 Free Radical-mediated Autooxidation of Tertiary Amines via the Aminium Radical Cation Intermediate</a></h4>
</td><td>

<h4 id="自由基介导的叔胺自然氧化反应胺自由基正离子中间体"><a href="#自由基介导的叔胺自然氧化反应胺自由基正离子中间体">3.5.3.3 自由基介导的叔胺自然氧化反应（胺自由基正离子中间体）</a></h4>
</td></tr>
<tr><td>

<p>In the presence of free radical sources, such as those produced by Udenfriend chemistry, tertiary amine drugs can also undergo autooxidation through the aminium radical cation intermediate.<span class="cite-ref"><sup>[117,118]</sup></span> During a formulation study of a potential therapeutic agent (<strong>X</strong>) for the treatment of stroke and severe head trauma, the drug candidate was found to autooxidize in an intravenous dosage form.<span class="cite-ref"><sup>[38]</sup></span> The liquid formulation contains a 10 mM, pH 4.5 lactate buffer. Various antioxidants, such as ascorbic acid, thioglycerol and sodium bisulfate, were tried to stabilize the formulation. Nevertheless, all these antioxidants were found actually to promote the oxidative degradation of the drug candidate. Based on these results as well as the structure of the drug candidate, it appears that the autooxidation is caused by the Udenfriend reaction. Although the original authors did not specify that the Udenfriend chemistry may be responsible for the observed instability of the formulated drug, the mechanism they proposed, in which a transition metal ion catalyzes the formation of reactive oxygen species from molecular oxygen and the oxidized metal ion is then recycled back to its reduced state by the antioxidants, largely resembles a typical Udenfriend pathway (see Section 3.2.1).</p>
</td><td>

<p>自由基条件下，比如 Udenfriend 体系中，叔胺可发生以胺自由基正离子为反应中间体的自然氧化反应。<span class="cite-ref"><sup>[117,118]</sup></span> 药物 <strong>X</strong> 是用于治疗中风和脑损伤的备选药物，在其制剂研究中发现，静脉注射剂中的药物分子可发生自然氧化。<span class="cite-ref"><sup>[38]</sup></span> 其制剂以 10 mM，pH 4.5 的乳酸缓冲溶液为基质，曾尝试添加诸如维生素C、硫甘油、亚硫酸钠等抗氧化剂以抑制其自然氧化作用，却适得其反，加入抗氧化剂反而加速了药物的氧化降解。结合药物分子结构，显而易见，其自然氧化可能是因为发生了 Udenfriend 反应。虽然原作者并没有确认 Udenfriend 反应是否与药物制剂的不稳定性有关，他所提出降解机理却完全可以看做是一个典型 Udenfriend 反应循环(详见小节 3.2.1)：过渡金属离子催化氧分子生成活性氧类，被氧化的金属离子经抗氧化剂还原回低价态。</p>
</td></tr>
<tr><td>

<p>The proposed mechanism is consistent with a ferrous ion spiking experiment they performed, in which the oxidative degradation of the drug candidate was accelerated. In this particular case, Udenfriend chemistry can operate in the following two ways. In the first scenario, the role of transition metal chelator can be played by the lactate buffer, which is a reasonably good iron chelator.<span class="cite-ref"><sup>[36]</sup></span> On the other hand, since the drug candidate itself contains a 2-hydroxyamine moiety, which is a good chelator for transition metal ions,<span class="cite-ref"><sup>[119]</sup></span> the authors surmised that it can directly chelate with the ferrous ion and the bound metal ion can therefore activate molecular oxygen. The reactive oxygen species formed can then extract an electron from the tertiary nitrogen of the drug candidate to produce the critical aminium radical cation as shown in Scheme 3.31.</p>
</td><td>

<p>此机理与下述实验结果相符：人为的加入铁离子可加速药物的氧化降解。在此例中，有两种方式构成 Udenfriend 反应。其一，乳酸作为螯合剂<span class="cite-ref"><sup>[36]</sup></span>与过渡金属离子络合；其二，此药物分子含有 β-羟基胺结构，也可充当过渡金属离子的螯合剂<span class="cite-ref"><sup>[119]</sup></span>，作者猜测此药物分子可与铁离子络合，而络合离子可活化氧分子生成活性氧类，随后从叔胺氮原子上夺取一个电子产生胺自由基正离子。详见 Scheme 3.31。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.31.png" alt="Scheme 3.31  " /><p class="caption"><span class="pic-ref">Scheme 3.31</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the above degradation mechanism, &quot;pathway a&quot; is proposed by the original authors, while &quot;pathway b&quot; is proposed by the current author. Both pathways involve the initial formation of the aminium radical cation intermediate. Both De La Mare<span class="cite-ref"><sup>[117]</sup></span> and Hong et al.<span class="cite-ref"><sup>[38]</sup></span> described this type of aminium radical cation and its conversion to the subsequent carbon-centered radical.</p>
</td><td>

<p>上述机理中，途径 a 是原作者提出的，途径 b 则由笔者提出。两种途径都涉及胺自由基正离子的生成。De La Mare<span class="cite-ref"><sup>[117]</sup></span> 和 Hong<span class="cite-ref"><sup>[38]</sup></span> 等人曾详尽描述了此种胺自由基正离子及其向碳自由基的转变。</p>
</td></tr>
<tr><td>

<h3 id="primary-and-secondary-amines"><a href="#primary-and-secondary-amines">3.5.4 Primary and Secondary Amines</a></h3>
</td><td>

<h3 id="伯胺与仲胺"><a href="#伯胺与仲胺">3.5.4 伯胺与仲胺</a></h3>
</td></tr>
<tr><td>

<p>In the case of primary and secondary amine oxidation, direct nucleophilic oxidation by hydrogen peroxide does not appear to be a significant degradation pathway, presumably because the nitrogen atoms in primary and secondary amines are less nucleophilic compared to tertiary amines. Aromatic primary and secondary amines are even less reactive. For example, excessive stress of cisapride by hydrogen peroxide at elevated temperature produced ~50% cisapride N-oxide, while no oxidation occurred on the aromatic primary amine moiety (Figure 3.8).<span class="cite-ref"><sup>[120]</sup></span></p>
</td><td>

<p>伯胺和仲胺的氧化降解途径中，直接亲核进攻过氧化氢而被氧化已不占主要。这可能是因为伯胺和仲胺的氮原子的亲核性远低于叔胺。芳香伯胺和仲胺的反应活性则更差。比如，以双氧水强制降解 cisapride，升高温度使之生成约 50% 的 N-氧化物，芳香伯胺却未见任何氧化(Figure 3.8)。<span class="cite-ref"><sup>[120]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.8.png" alt="Figure 3.8   Cisapride 在双氧水强制降解实验中的 N-氧化位点。 N-Oxidation site of cisapride under stress by hydrogen peroxide." /><p class="caption"><span class="pic-ref">Figure 3.8</span>   Cisapride 在双氧水强制降解实验中的 N-氧化位点。<br /> N-Oxidation site of cisapride under stress by hydrogen peroxide.</p>
</div>
</td></tr>
<tr><td>

<p>On the other hand, hydrogen peroxide can be &quot;activated&quot; by interacting with certain components in a formulation or in a stress system as discussed above in Section 3.3.1 to form peracids, acetonitrile adducts, and peroxymono-carbonates, respectively. The resulting species are more reactive than hydrogen peroxide. For example, peracids are capable of reacting with primary and secondary amines to yield initially hydroxylamines<span class="cite-ref"><sup>[121,122]</sup></span> which are usually subject to further degradation depending upon the structure of the amines (Scheme 3.32).<span class="cite-ref"><sup>[123,124]</sup></span></p>
</td><td>

<p>然而，如小节 3.3.1 中所述，过氧化氢可被制剂中或强制降解实验体系中的某些特定组分“活化”，生成过氧酸、腈类加成物、过一碳酸盐等。相比于过氧化氢，这些活化形态的反应活性更高。比如，过氧酸可与伯胺或仲胺生成羟胺<span class="cite-ref"><sup>[121,122]</sup></span>，而是否会进一步降解则要取决于其结构(Scheme 3.32)。<span class="cite-ref"><sup>[123,124]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.32.png" alt="Scheme 3.32  " /><p class="caption"><span class="pic-ref">Scheme 3.32</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Scheme 3.32 shows that the initial degradation product formed is N-oxide, which immediately isomerizes to N-hydroxylamine.<span class="cite-ref"><sup>[125]</sup></span> This isomerization can be viewed as a special case of the Meisenheimer rearrangement, in which the proton rearranges from the nitrogen to the oxygen. The N-hydroxylamine formed can further degrade to imine if one of the alkyl groups contains an α-H.</p>
</td><td>

<p>Scheme 3.32 显示，首先形成 N-氧化物，但立即异构化为羟胺。<span class="cite-ref"><sup>[125]</sup></span> 此异构化可看作是 Meisenheimer 重排的一个特例：质子从氮原子迁移到了氧原子上。若烷基存在 α-H，羟胺可进一步降解生成亚胺。</p>
</td></tr>
<tr><td>

<p>The imine can further oxidize to become a nitrone. All the synthetically useful procedures for preparing a nitrone from secondary amine use catalysts to assist the oxidation by hydrogen peroxide.<span class="cite-ref"><sup>[126]</sup></span> Hence, how meaningful the pathway is for secondary amines in autooxidation is questionable. On the other hand, the imine formed is an electrophile which is susceptible to nucleophilic attack. When the nucleophile is water or hydroxide, hydrolysis of the imine can occur.</p>
</td><td>

<p>亚胺进一步氧化则生成硝酮(nitrone)。而在合成化学中制备硝酮的方法恰是催化剂辅助的过氧化氢氧化仲胺。<span class="cite-ref"><sup>[126]</sup></span> 然而，此种机理在仲胺的自然氧化中的意义仍有待商榷。另一方面，亚胺也容易受亲核进攻，而亲核剂可以是水、羟基化物或亚胺的水解产物。</p>
</td></tr>
<tr><td>

<p>In the presence of radical species, the initial oxidation product of a secondary amine would be a hydroxylamine radical according to a recent study using electron spin resonance (ESR) and theoretical calculation (Scheme 3.33).<span class="cite-ref"><sup>[127]</sup></span></p>
</td><td>

<p>自由基条件下，仲胺的初始氧化产物为羟胺自由基。详见最新的电子自旋共振(ESR)和理论计算研究(Scheme 3.33)。<span class="cite-ref"><sup>[127]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.33.png" alt="Scheme 3.33   Reproduction from Reference 127 with permission." /><p class="caption"><span class="pic-ref">Scheme 3.33</span>   Reproduction from Reference 127 with permission.</p>
</div>
</td></tr>
<tr><td>

<p>The secondary hydroxylamine radical formed can obviously abstract a hydrogen to yield the corresponding hydroxylamine. In general, secondary hydroxylamines are usually not very stable and hence difficult to isolate. There are a few exceptions, for example, N-hydroxydesloratadine, which is an oxidative degradant of desloratadine and is stable enough to be isolated.<span class="cite-ref"><sup>[128]</sup></span> In the case of a stable free radical species, TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl) (Figure 3.9), the presence of the surrounding 2,2,6,6-tetramethyl groups stabilizes the free radical on the secondary amine.</p>
</td><td>

<p>生成的羟胺自由基可夺氢生成相应的羟胺。一般来说，羟胺不是很稳定，故而难以被分离。但也有例外，比如 desloratadine 的氧化降解产物 N-hydroxydesloratadine，足够稳定可被纯化分离。<span class="cite-ref"><sup>[128]</sup></span> 也有能稳定存在的自由基，比如 TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl)，其结构式见 图 3.9，四个甲基的存在稳定了这个自由基。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.9.png" alt="Figure 3.9   TEMPO 的结构。Structure of TEMPO." /><p class="caption"><span class="pic-ref">Figure 3.9</span>   TEMPO 的结构。<br />Structure of TEMPO.</p>
</div>
</td></tr>
<tr><td>

<p>TEMPO has been extensively used in ESR and nuclear magnetic resonance (NMR) studies as a stable free radical label<span class="cite-ref"><sup>[129,130]</sup></span> and a catalyst for oxidation of alcohols.<span class="cite-ref"><sup>[131,132]</sup></span></p>
</td><td>

<p>TEMPO 作为稳定的自由基标记物，被广泛应用于 ESR 和核磁共振(NMR)。<span class="cite-ref"><sup>[129,130]</sup></span> 也作为催化剂催化醇的氧化反应。<span class="cite-ref"><sup>[131,132]</sup></span></p>
</td></tr>
<tr><td>

<h3 id="enamines-and-imines-schiff-bases"><a href="#enamines-and-imines-schiff-bases">3.5.5 Enamines and Imines (Schiff Bases)</a></h3>
</td><td>

<h3 id="烯胺和亚胺希夫碱"><a href="#烯胺和亚胺希夫碱">3.5.5 烯胺和亚胺(希夫碱)</a></h3>
</td></tr>
<tr><td>

<p>Enamines and imines are interconvertable species through tautomerization. Nevertheless, they have quite different reactivities: enamines are nucleophiles while imines are electrophiles. Imines are formed by condensation between primary amines and aldehydes; replacement of the primary amines by secondary amines will produce iminium salts. The condensation reaction is usually reversible except for a few cases where the resulting imines may be stabilized by additional structural features such as conjugation and/or cyclization. Both imines and iminium salts can tautomerize to enamines. The above processes can be summarized in Scheme 3.34.</p>
</td><td>

<p>烯胺和亚胺是一对互变异构体。但两者的反应性差异巨大：烯胺是亲核试剂而亚胺是亲电试剂。伯胺与醛缩合生成亚胺，若是仲胺则生成亚胺离子。此缩合反应一般是可逆的，但一些特例中，所形成亚胺因共轭或环化而得以稳定存在。此转化关系总结于 Scheme 3.34。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.34.png" alt="Scheme 3.34  " /><p class="caption"><span class="pic-ref">Scheme 3.34</span>  </p>
</div>
</td></tr>
<tr><td>

<p>According to the work by Malhotra et al., enamines and imines of α,β-unsaturated ketones are susceptible to autooxidation at the γ-position, leading to the formation of 1,4-diones.<span class="cite-ref"><sup>[133]</sup></span> Malhotra et al. proposed, using the pyrrolidine enamine of 10-methyl-Δ<sup>1(9)</sup>-octalone-2 as the model compound, that the enamine of an α,β-unsaturated ketone can directly transfer an electron from the nitrogen to molecular oxygen in the initiation step of a free radical-mediated oxidation. The aminium radical cation intermediate formed, which very much resembles the one generated from tertiary amines during the free radical-mediated autooxidation of the latter (Section 3.5.3.3), then reacts with molecular oxygen at the γ-position to give the γ-peroxy radical. The latter can abstract a H to form the γ-peroxide, which ultimately yields the corresponding 1,4-dione (Scheme 3.35).</p>
</td><td>

<p>参考 Malhotra 等人的工作，α,β-不饱和酮的烯胺和希夫碱容易在γ位发生自然氧化，生成 1,4-二酮。<span class="cite-ref"><sup>[133]</sup></span> 以 10-methyl-Δ<sup>1(9)</sup>-octalone-2 为模型化合物，Malhotra 等人认为，在自由基条件下，此不饱和酮可传递电子给氧分子，随之生成胺自由基正离子（叔胺受自由基氧化反应有可能生成胺自由基正离子，详见小节 3.5.3.3），与氧分子反应于γ位生成过氧自由基。过氧自由基夺氢成为过氧化物，最终生成 1,4-二酮(Scheme 3.35)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.35.png" alt="Scheme 3.35  " /><p class="caption"><span class="pic-ref">Scheme 3.35</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The authors also observed a striking catalytic effect by transition metal ions such as Cu<sup>2+</sup> and Fe<sup>3+</sup>, and attributed it to their ability to accept an electron from the enamine in the initiation step. Such results may imply that, even in cases where no metal ions were purposely added, the initiation step may be catalyzed by trace levels of metal ions, rather than proceed by direct reaction between the enamine and molecular oxygen as shown in Scheme 3.35. Imines are susceptible to the same autooxidation once they tautomerize to the corresponding enamine forms.</p>
</td><td>

<p>此外，原作者还观测到了过渡金属离子，比如 Cu<sup>2+</sup> 和 Fe<sup>3+</sup> 有显著的催化作用，这是因为这些离子可在反应引发阶段从烯胺那里夺取电子。这些结果似乎暗示了，倘若不人为地添加金属离子，此反应也可被体系中痕量的金属离子所引发；似乎不会出现 Scheme 3.35 中描述的烯胺和分子氧的直接反应。亚胺互变异构为相应的烯胺时，同样会发生类似的自然氧化。</p>
</td></tr>
<tr><td>

<p>In a stability study of tetrazepam, it was proposed that one of the major degradants, tetrazepam 3'-ketone, formed via the pathway outlined in Scheme 3.35, after the embedded imine of α,β-unsaturated ketone tautamerizes to the enamine form.<span class="cite-ref"><sup>[103]</sup></span> The imine moiety remained intact during this particular degradation pathway, apparently due to the stabilization provided by the seven-membered ring of benzodiazepan and conjugation to the phenyl moiety (Scheme 3.36).</p>
</td><td>

<p>Tetrazepam 的稳定性研究中，曾提出主要讲解产物之一的 tetrazepam 3'-ketone 是经历了 Scheme 3.35 中所示的机理而生成的：α,β-不饱和酮的希夫碱互变异构为烯胺，然后发生了γ位的自然氧化。<span class="cite-ref"><sup>[103]</sup></span> 亚胺结构在讲解过程中得以幸存应归功于二氮䓬七元环和苯环的稳定作用(Scheme 3.36)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.36.png" alt="Scheme 3.36  " /><p class="caption"><span class="pic-ref">Scheme 3.36</span>  </p>
</div>
</td></tr>
<tr><td>

<h3 id="thioethers-organic-sulfides-sulfoxides-thiols-and-related-species"><a href="#thioethers-organic-sulfides-sulfoxides-thiols-and-related-species">3.5.6 Thioethers (Organic Sulfides), Sulfoxides, Thiols and Related Species</a></h3>
</td><td>

<h3 id="硫醚有机硫化物亚砜硫醇以及相关物类"><a href="#硫醚有机硫化物亚砜硫醇以及相关物类">3.5.6 硫醚(有机硫化物)，亚砜，硫醇以及相关物类</a></h3>
</td></tr>
<tr><td>

<p>One of the most common degradation pathways of the thioether moiety in drug molecules is probably the oxidation caused by hydroperoxides present in certain excipients such as PEG.<span class="cite-ref"><sup>[41]</sup></span> This oxidation can take place via nucleophilic attack by the sulfur on the hydroperoxide.<span class="cite-ref"><sup>[134,135]</sup></span> From the perspective of the hydroperoxide, one of its two oxygens is electrophilically transferred to the nucleophilic sulfur. Hence, this process (nucleophilic oxidation of sulfur) is also called &quot;electrophilic oxygen transfer&quot;, which is similar to the epoxidation of electron-rich olefins by hydroperoxides (see Scheme 3.13). On the other hand, the hydroperoxide oxygen can also transfer to electrophilic oxidation substrates via a &quot;nucleophilic oxygen transfer&quot; process, which is represented by the well-known Baeyer-Villiger oxidation of ketones and aldehydes.<span class="cite-ref"><sup>[136]</sup></span> Since sulfoxides are both nucleophilic (due to the lone electron pair on the sulfur) and electrophilic, they can be oxidized via both the &quot;electrophilic oxygen transfer&quot; and &quot;nucleophilic oxygen transfer&quot; processes.<span class="cite-ref"><sup>[137]</sup></span></p>
</td><td>

<p>药物分子中的硫醚结构最常见的降解途径是被某些赋形剂（比如 PEG）中存在的过氧化物所氧化。<span class="cite-ref"><sup>[41]</sup></span> 这种氧化一般是硫原子亲核进攻过氧化物。<span class="cite-ref"><sup>[134,135]</sup></span> 以过氧化氢来看，它的两个氧原子皆可迁移到亲核性的硫原子上。此过程（硫原子亲核进攻被氧化）称为“亲电氧转移”，恰类似于富电子烯烃与过氧化物发生的环氧化反应（见 Scheme 3.13）。此外，过氧化氢的氧原子可通过所谓的“亲核氧转移”机理与亲电性的底物反应，此反应知名度颇高，即醛酮的 Baeyer-Villiger 氧化反应。<span class="cite-ref"><sup>[136]</sup></span> 由于亚砜既可做亲核试剂（硫原子上存在孤对电子）也可做亲电试剂，以上两种“氧转移”机理都可以氧化亚砜。<span class="cite-ref"><sup>[137]</sup></span></p>
</td></tr>
<tr><td>

<p>In other cases, the thioether moiety can undergo autooxidation through one of the following two mechanisms: photochemical oxidation by singlet molecular oxygen (discussed in Chapter 6) and free radical-mediated autooxidation initiated by peroxyl radicals. The latter mechanism was proposed to proceed through a unique 2σ/1σ* three-electron-bonded disulfide radical cation, which can then react with either superoxide anion radical or water and molecular oxygen (Scheme 3.37).<span class="cite-ref"><sup>[138-141]</sup></span></p>
</td><td>

<p>硫醚的氧化可经由一下两种机理：与单线态氧发生光化学氧化（将于第六章讨论）和过氧自由基引发的自由基氧化。一般认为自由基氧化将经历独特的 2σ/1σ* 三电子键键合的二硫化物自由基正离子( 2σ/1σ* three-electron-bonded disulfide radical cation )，它将与超氧阴离子自由基、水或分子氧进一步反应(Scheme 3.37)。<span class="cite-ref"><sup>[138-141]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.37.png" alt="Scheme 3.37  " /><p class="caption"><span class="pic-ref">Scheme 3.37</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Alternately, other species containing nucleophilic hetero atoms (X) such as oxygen and nitrogen can replace the second molecule of the thioether (in the second step of Scheme 3.37) in the formation of a similar 2σ/1σ* three-electron-bonded S-X radical cation.<span class="cite-ref"><sup>[141]</sup></span></p>
</td><td>

<p>其他含有亲核性杂原子(X)的物类，比如氧气分子或氮气分子，可代替硫醚分子（Scheme 3.37 第二步反应）生成类似的 2σ/1σ* 三电子键 S-X 键合自由基正离子。<span class="cite-ref"><sup>[141]</sup></span></p>
</td></tr>
<tr><td>

<p>This mechanism seems to be consistent with several observations made during the study of the free radical-mediated formation of sulfoxides:<span class="cite-ref"><sup>[128]</sup></span> first, the yield from sulfoxide formation increases with increasing pH, probably due to the facilitation of the deprotonation step at higher pH. Second, it has been demonstrated that the oxygen on the sulfoxide originates from water rather than molecular oxygen, although the presence of the latter significantly enhances the sulfoxide yield. This observation is different from the formation of sulfoxide via nucleophilic oxidation by hydroperoxide (see also Section 3.3.1), where the sulfoxide oxygen comes from the hydroperoxide. Third, the yield of sulfoxide correlates with the concentration of the oxidation substrate, thio-ether. The sulfoxide formed via either the radical or non-radical-mediated pathways is reasonably stable and can be isolated, although it can be further oxidized to form sulfone upon excessive oxidation.</p>
</td><td>

<p>此机理与自由基反应制备亚砜的研究中所观察到的现象相符<span class="cite-ref"><sup>[128]</sup></span>：首先，升高 pH 可提高亚砜的产率，这可能是因为高 pH 使得去质子化更容易。其次，证实了迁移到硫原子上的氧来自于水分子而非氧气，但氧气的存在却能明显提高产率。此点不同于亲核进攻过氧化物而生成亚砜——此氧原子来自于过氧化物。再次，亚砜的产率与底物硫醚的浓度息息相关。无论是经历自由基机理还是非自由基机理，所生成亚砜都很稳定，足以被分离出来。但在过度氧化中却也可以被进一步氧化。</p>
</td></tr>
<tr><td>

<p>As we have discussed so far, pH can have an impact on the oxidation of thioethers and sulfoxides under both the radical and non-radical-mediated conditions. Under the former condition, we have just shown that oxidation is facilitated at higher pH. Under non-radical conditions (electrophilic and nucleophilic oxygen transfers), electrophilic oxygen transfer is favored at lower pH, while nucleophilic oxygen transfer is favored at higher pH. The oxidation of amines and related molecules is enhanced at higher pH, regardless of radical or non-radical-mediated conditions (see Section 3.5.3). One may take advantage of such differences in formulation development by selecting an optimal pH where the degradation of the drug candidate would be minimal.</p>
</td><td>

<p>至此，可以看到，无论是在自由基条件或非自由基条件下，pH 都会影响硫醚和亚砜的氧化行为。前文中我们提到自由基机理下，高 pH 使氧化更容易发生。但在非自由基条件下，低 pH 有利于发生亲电氧转移，高 pH 则有利于亲核氧转移。而胺及其相关化合物，无论自由基或非自由基条件下，都是高 pH 有利于氧化反应发生。在制剂研究中应巧妙利用此中差异，选定最佳的 pH 使得药物的降解速率最慢。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.38.png" alt="Scheme 3.38  " /><p class="caption"><span class="pic-ref">Scheme 3.38</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In a particular oxidative degradation case involving thioethers and sulfoxides, it may be difficult to tell whether a radical or non-radical mechanism is involved. Sometimes, both mechanisms may be operative. The oxidative degradation mechanism of methionine has been studied quite extensively, owing to its relevance to protein degradation as a protein amino acid.<span class="cite-ref"><sup>[142-149]</sup></span> Oxidation of methionine to the corresponding sulfoxide (Scheme 3.38) can be either via the nucleophilic (non-radical) pathway, which would be straightforward, as discussed in Section 3.3.1, or via the radical-mediated pathway which should follow the mechanism illustrated in Scheme 3.37.</p>
</td><td>

<p>对于某个特定的降解案例，很难判断硫醚或亚砜是否经历了自由基机理。有时，自由基机理和非自由基机理共存。甲硫氨酸的氧化降解因其在多肽降解中的重要地位而被广泛研究。<span class="cite-ref"><sup>[142-149]</sup></span> 甲硫氨酸被氧化为相应的亚砜(Scheme 3.38)，即可如小节 3.3.1 所介绍的那样直接与过氧化物反应（非自由基机理），也可以是 Scheme 3.37 所示的自由基氧化机理。</p>
</td></tr>
<tr><td>

<p>Several drug molecules containing aliphatic thioether moieties that undergo the same S-oxidation include montelukast sodium,<span class="cite-ref"><sup>[150,151]</sup></span> cimetidine,<span class="cite-ref"><sup>[152-154]</sup></span> and ranitidine (Figure 3.10).<span class="cite-ref"><sup>[155,156]</sup></span></p>
</td><td>

<p>含有脂肪族硫醚结构的药物分子比如 montelukast sodium<span class="cite-ref"><sup>[150,151]</sup></span>、cimetidine<span class="cite-ref"><sup>[152-154]</sup></span>、ranitidine (Figure 3.10) 都可发生硫原子的氧化，生成相应的亚砜。<span class="cite-ref"><sup>[155,156]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.10.png" alt="Figure 3.10   montelukast sodium、cimetidine、ranitidine 的结构。 Structures of montelukast sodium, cimetidine, and ranitidine." /><p class="caption"><span class="pic-ref">Figure 3.10</span>   montelukast sodium、cimetidine、ranitidine 的结构。<br /> Structures of montelukast sodium, cimetidine, and ranitidine.</p>
</div>
</td></tr>
<tr><td>

<p>Since one alkyl group that attaches to the sulfur is chiral in montelukast sodium, the resulting two sulfoxides are diastereomers with respect to each other. In the cases of cimetidine and ranitidine where all the alkyl groups are achiral, the resulting sulfoxides are enantiomers in each case.</p>
</td><td>

<p>亚砜的硫原子所连接的两个基团不相同时，会产生手性。Montelukast sodium 中与硫原子键合的烷基含有手性碳，故而所生成的亚砜是一对非对映异构体。Cimetidine 和 ranitidine 没有手性基团，所生成的亚砜则是对应异构体。</p>
</td></tr>
<tr><td>

<p>The mechanisms discussed above are based on the studies of aliphatic thioethers. For aryl thioethers, it appears that the non-radical, nucleophilic oxidation mechanism can be readily applied. For example, oxidation of the diaryl thioether moiety of phenothiazine-based drugs (Figure 3.11; see also Section 3.5.3.1) by hydroperoxides at room temperature should proceed via the nucleophilic oxidation mechanism.<span class="cite-ref"><sup>[98]</sup></span> In another case where an experimental diaryl thioether drug was formulated using BHT as the antioxidant, Puz et al. found that the use of 2% BHT in the tablet coating was able to suppress the majority of the sulfur oxidation in two accelerated stability studies (40 °C/75% RH and 50 °C/20% RH).<span class="cite-ref"><sup>[157]</sup></span> Since BHT exerts its anti-oxidation effect by inhibiting the free radical propagation step in a radical-mediated autooxida-tion, it appears that a free radical mechanism is mostly likely to be responsible for the sulfoxide formation in this experimental formula under the above accelerated stability conditions.</p>
</td><td>

<p>以上所讨论都是脂肪族硫醚。若换做是芳香族硫醚，非自由基机理和亲核氧化机理都有可能出现。例如，室温下，过氧化物氧化吩噻嗪类药物(Figure 3.11，另见小节 3.5.3.1)中的二芳基硫醚结构，是经历的亲核氧化机理。<span class="cite-ref"><sup>[98]</sup></span> 又比如，某含有二芳基硫醚结构的备选药物，在制剂中加入 BHT 作为抗氧化剂。Puz 等人发现，在片剂包衣中加入 2% 的 BHT 可明显抑制硫原子被氧化（加速稳定性实验40 °C/75% RH 和 50 °C/20% RH 两条件下，成效显著）。<span class="cite-ref"><sup>[157]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.11.png" alt="Figure 3.11   吩噻嗪类药物和另一实验性药物分子中二芳基硫醚结构的氧化，箭头指示了氧化位置。 Oxidation of the diaryl thioether moiety of phenothiazine-based drugs and an experimental drug. The arrows indicate the S-oxidation sites." /><p class="caption"><span class="pic-ref">Figure 3.11</span>   吩噻嗪类药物和另一实验性药物分子中二芳基硫醚结构的氧化，箭头指示了氧化位置。<br /> Oxidation of the diaryl thioether moiety of phenothiazine-based drugs and an experimental drug. The arrows indicate the S-oxidation sites.</p>
</div>
</td></tr>
<tr><td>

<p>Very few small molecule drugs contain the thiol (sulfhydro) functionality. Two notable examples are captopril, a first generation angiotensin-converting enzyme (ACE) inhibitor which is still used clinically for the treatment of hypertension, and N-acetylcysteine, a drug mainly used as a mucolytic agent. The main degradants of both compounds are the corresponding dimers (captopril disulfide and N-acetylcysteine disulfide) formed through the oxidative coupling of the two thiol groups.<span class="cite-ref"><sup>[158-160]</sup></span> The degradation pathways of the thiol functional group will be further discussed with the amino acid cysteine in Chapter 7, Chemical Degradation of Biological Drugs.</p>
</td><td>

<p>少数小分子药物中包含硫醇结构（硫羟基）。有两个例子不得不提，一个是第一代血管紧张肽转化酶(angiotensin-converting enzyme, ACE)抑制剂 captopril，至今仍用于高血压的临床治疗。另一则是溶粘蛋白剂 N-acetylcysteine。这两个化合物的的硫羟基发生氧化偶联形成相应的二聚体（captopril disulfide 和 N-acetylcysteine disulfide）。<span class="cite-ref"><sup>[158-160]</sup></span> 硫羟基的此种降解途径将在第七章结合半胱氨酸做进一步讨论。</p>
</td></tr>
<tr><td>

<h3 id="examples-of-carbanionenolate-mediated-autooxidation"><a href="#examples-of-carbanionenolate-mediated-autooxidation">3.5.7 Examples of Carbanion/enolate-mediated Autooxidation</a></h3>
</td><td>

<h3 id="碳正离子烯醇负离子介导的自然氧化的实例"><a href="#碳正离子烯醇负离子介导的自然氧化的实例">3.5.7 碳正离子/烯醇负离子介导的自然氧化的实例</a></h3>
</td></tr>
<tr><td>

<p>As discussed in Section 3.4, much less is known about carbanion/enolate-mediated (or base-catalyzed) autooxidation compared to free radical-mediated autooxidation. This is primarily due to the fact that free radicals, in particular peroxy radical, mediate the majority of autooxidative degradation observed in drug substances and drug formulations. Nevertheless, for a group of compounds that contain an acidic CH<sub>n</sub> (n is usually 1 to 2), carbanion/enolate-mediated autooxidation may be the predominant process, especially in solutions or liquid formulations where pH is near neutral or alkaline conditions.</p>
</td><td>

<p>如同小节 3.4 中已经提到的那样，较之于自由基介导的自然氧化，对碳正离子/烯醇负离子介导（或碱催化）的自然氧化仍知之甚少。毕竟在原料药和制剂中观测到的自然氧化大部分是自由基参与的，而这一般会是过氧自由基。但是，部分含有酸性 CH<sub>n</sub> (n = 1, 2)化合物的自然氧化中，碳正离子/烯醇负离子介导的自然氧化一般是主要降解途径，尤其是在溶液（或制剂溶液）pH 为中性或碱性时。</p>
</td></tr>
<tr><td>

<p>During a study of ketorolac tromethamine stability in aqueous buffers with a wide range of pH under elevated temperatures (60-100 °C), the formation of the initial major degradant at pH &gt; 4.8 was attributed to the carbanion-mediated autooxidation as shown in Scheme 3.39.<span class="cite-ref"><sup>[161]</sup></span></p>
</td><td>

<p>在研究 ketorolac tromethamine 在不同温度下，不同 pH 的缓冲溶液中的稳定性时发现，pH &gt; 4.8 时，初始主要降解产物即通过碳正离子/烯醇负离子介导的自然氧化生成。此反应见 Scheme 3.39。<span class="cite-ref"><sup>[161]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.39.png" alt="Scheme 3.39  " /><p class="caption"><span class="pic-ref">Scheme 3.39</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In this proposed mechanism, the formation of the carbanion on the position α to the carboxyl group and the subsequent reaction with molecular oxygen precedes the decarboxylation step. The possibility of an alternate mechanism where initial decarboxylation is followed by reaction with molecular oxygen was excluded, based on the absence of a particular degradant (the decarboxylated degradant) which would be formed under the alternate degradation mechanism.</p>
</td><td>

<p>在上述机理中，于羰基 α 位形成碳正离子，随即与氧分子反应，最终脱羧。另一条途径，先脱羧而后与氧分子反应，虽看似合理，但并未观测到关键的脱羧降解产物故而可以排除。而脱羧在其他降解机理中的确可以发生。</p>
</td></tr>
<tr><td>

<p>Another good example of carbanion/enolate-mediated autooxidation can be found in the case of rofecoxib. The autooxidation of rofecoxib was originally hypothesized as being mediated by free radicals.<span class="cite-ref"><sup>[162]</sup></span> Harmon et al. found that this autooxidation proceeds nearly two orders of magnitude faster than a peroxy free radical-mediated autooxidation and the stress condition lacks an obvious source of the free radical.<span class="cite-ref"><sup>[8]</sup></span> Hence, a systematic mechanistic study was undertaken which provided convincing evidence that the autooxidation of rofecoxib is mediated by the rofecoxib carbanion/enolate which is readily generated under alkaline conditions (Scheme 3.40).</p>
</td><td>

<p>另一个碳正离子/烯醇负离子介导的自然氧化的实例是 rofecoxib。其降解机理最初被认为是自由基氧化。<span class="cite-ref"><sup>[162]</sup></span> 但 Harmon 等人发现在明显缺少自由基来源的强制降解条件下，其自然氧化速度极快，比过氧自由基介导的自然氧化快约 100 倍。<span class="cite-ref"><sup>[8]</sup></span> 为此，Harmon 等人对其降解机理进行了系统研究，最终以充足的证据证明了 rofecoxib 在碱性条件下很容易生成相应的碳正离子/烯醇负离子，从而迅速发生自然氧化(Scheme 3.40)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.40.png" alt="Scheme 3.40  " /><p class="caption"><span class="pic-ref">Scheme 3.40</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Reddy and Corey performed a similar base-catalyzed autooxidation study of rofecoxib at about the same time;<span class="cite-ref"><sup>[163]</sup></span> and although their reaction medium was a mixture of tetrahydrofuran (THF) and water with one equivalent LiOH, which is different from the mixture of acetonitrile and phosphate buffer (pH 11 or 12) used by Harmon et al.,<span class="cite-ref"><sup>[8]</sup></span> the same final degradants were observed. In both cases, rofecoxib anhydride was found to exist in appreciable quantities when the organic co-solvent is present in high percentage. Since Harmon et al. used high-performance liquid chromatography (HPLC) to monitor the autooxidation reaction, they were able to observe the transient rofecoxib hydroperoxide intermediate.<span class="cite-ref"><sup>[8]</sup></span> When treated with triphenylphosphine, the hydroperoxide was converted to rofecoxib γ-hydroxybutenolide.</p>
</td><td>

<p>与之同时，Reddy 和 Corey 对 rofecoxib 进行类似的碱催化自然氧化研究<span class="cite-ref"><sup>[163]</sup></span>，但他们使用的溶剂是四氢呋喃/水混合溶液，等当量的 LiOH。这不同于 Harmon 等人所使用的乙腈/磷酸盐缓冲液(pH = 11 或 12)<span class="cite-ref"><sup>[8]</sup></span>。但两方都观测到了相同的降解产物，且当有机溶剂比例较高时，都发现了数量可观的 rofecoxib anhydride。Harmon 等人使用 HPLC 监测到了倏忽即逝的反应中间体 rofecoxib hydroperoxide。<span class="cite-ref"><sup>[8]</sup></span> 在反应体系中加入三苯基膦，此氢过氧化物转化为 rofecoxib γ-hydroxybutenolide。</p>
</td></tr>
<tr><td>

<p>Most degradation of corticosteroidal drugs containing a 1,3-dihydroxy-acetone side chain such as hydrocortisone, betamethasone and dexamethasone, is oxidative in nature, occurring at the D-ring which contains this side chain.<span class="cite-ref"><sup>[83,164,165]</sup></span> Edmonds et al. studied autooxidative degradation of dexamethasone in neutral to alkaline aqueous buffers.<span class="cite-ref"><sup>[7]</sup></span> At pH 7.4 and room temperature, autooxidation of dexamethasone proceeded reasonably fast: it decomposed completely in 28 days to give dexamethasone glyoxal (dexamethasone 21-aldehyde). With increased pH, the autooxidation proceeded much faster and further degradants such as dexamethasone etioacid (dexa-methasone 17-acid) and dexamethasone glycolic acid were formed. Li et al. performed a detailed mechanistic study of the base-catalyzed autooxidation of betamethasone with high resolution LC-MS and <sup>18</sup>O<sub>2</sub>, from which the degradation pathway in Scheme 3.41 was proposed.<span class="cite-ref"><sup>[82]</sup></span></p>
</td><td>

<p>大多数皮质类固醇药物，比如 hydrocortisone、betamethasone 和 dexamethasone，带有侧链的 D 环会发生氧化降解。<span class="cite-ref"><sup>[83,164,165]</sup></span> Edmonds 等人研究了中性和碱性缓冲溶液中 dexamethasone 自然氧化降解。<span class="cite-ref"><sup>[7]</sup></span> 室温下，pH 7.4，自然氧化进行较快：28 天完全分解得到 dexamethasone glyoxal (dexamethasone 21-aldehyde)。进一步提高 pH，则反应加速且发现了后续降解产物如 dexamethasone etioacid (dexa-methasone 17-acid) 和 dexamethasone glycolic acid。Li 等人使用 LC-MS 和同位素标记的 <sup>18</sup>O<sub>2</sub>，对此碱催化 betamethasone 自然氧化反应的机理进行了研究，提出了 Scheme 3.41 所示降解机理。<span class="cite-ref"><sup>[82]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.41.png" alt="Scheme 3.41  " /><p class="caption"><span class="pic-ref">Scheme 3.41</span>  </p>
</div>
</td></tr>
<tr><td>

<p>It appears that this mechanism can explain the majority of the oxidative degradation behaviors of the corticosteroids containing a 1,3-dihydroxyacetone side chain which include not only betamethasone but also hydrocortisone,<span class="cite-ref"><sup>[164]</sup></span> prednisolone,<span class="cite-ref"><sup>[165]</sup></span> and dexamethasone.<span class="cite-ref"><sup>[7,166]</sup></span> In a study of dexamethasone in an aqueous ophthalmic suspension,<span class="cite-ref"><sup>[166]</sup></span> dexamethasone 17-formyloxy-17-acid was isolated as one of the degradants. In addition, the dexamethasone anhydride intermediate, analogous to constituent (<strong>9</strong>) in Scheme 3.41, was also implicated. These results provide further support for the general applicability of the mechanism proposed in Scheme 3.41 for this class of corticosteroids.</p>
</td><td>

<p>此机理似乎完美解释了以 1,3-二羟基丙酮为侧链的皮质类固醇的氧化降解行为，不仅是 betamethasone，hydrocortisone<span class="cite-ref"><sup>[164]</sup></span>、prednisolone<span class="cite-ref"><sup>[165]</sup></span>、dexamethasone<span class="cite-ref"><sup>[7,166]</sup></span> 也在其列。在 dexamethasone 的某眼用水混悬液制剂中<span class="cite-ref"><sup>[166]</sup></span>，分离出了降解产物 dexamethasone 17-formyloxy-17-acid。且发现体系中还存在dexamethasone anhydride 和化合物 <strong>9</strong>。这些结果进一步肯定了此机理的普适性。</p>
</td></tr>
<tr><td>

<p>In drug molecules where a more acidic CH<sub>n</sub> is present, carbanion/enolate-mediated autooxidation can take place spontaneously. For example, phenylbutazone undergoes very rapid autooxidation on a silica gel thin-layer chromatography (TLC) plate to produce two major degradants, 4-hydroxyphenylbutazone and N-(a-ketocaproyl)hydrozobenzene.<span class="cite-ref"><sup>[167]</sup></span> This facile autooxidation may be better explained by the mechanism shown in Scheme 3.42, which is different from the one proposed during the original study.</p>
</td><td>

<p>若药物分子中存在更多的酸性 CH<sub>n</sub>，碳正离子/烯醇负离子介导的自然氧化反应甚至可以自发进行。例如，phenylbutazone 在硅胶薄层色谱板(TLC)上会迅速发生自然氧化产生两个主要降解产物：4-hydroxyphenylbutazone 和 N-(a-ketocaproyl)hydrozobenzene。<span class="cite-ref"><sup>[167]</sup></span> Scheme 3.42 所示机理可以很好的解释此化合物为何对氧化如此敏感，原作者所提出的机理与此略有不同。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.42.png" alt="Scheme 3.42  " /><p class="caption"><span class="pic-ref">Scheme 3.42</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In solid dosage formulations of phenylbutazone containing anti-acid ingredients, phenylbutazone was found to undergo significant oxidative and hydrolytic degradation. This degradation behavior is consistent with a base-catalyzed autooxidation mechanism.</p>
</td><td>

<p>Phenylbutazone 的固体制剂中若含有抗酸成分，phenylbutazone 将发生显著的氧化和水解降解。其降解行为与碱催化自然氧化机理吻合。</p>
</td></tr>
<tr><td>

<h3 id="oxidation-of-drugs-containing-alcohol-aldehyde-and-ketone-functionalities"><a href="#oxidation-of-drugs-containing-alcohol-aldehyde-and-ketone-functionalities">3.5.8 Oxidation of Drugs Containing Alcohol, Aldehyde, and Ketone Functionalities</a></h3>
</td><td>

<h3 id="含有醇醛酮官能团的药物分子的氧化"><a href="#含有醇醛酮官能团的药物分子的氧化">3.5.8 含有醇、醛、酮官能团的药物分子的氧化</a></h3>
</td></tr>
<tr><td>

<p>Oxidation of primary alcohols yields aldehydes, while oxidation of secondary alcohols produces ketones. Autooxidation of both primary and secondary alcohols is likely to proceed via a free radical-mediated mechanism. Williams et al. performed an electron paramagnetic resonance (EPR) study on the autooxidation of an amorphous hydroxyl-containing drug substance in which two radical species were detected and can be attributed, respectively, to a carbon-centered radical and a peroxide radical.<span class="cite-ref"><sup>[168]</sup></span> The bond dissociation energies (BDE) of the C-H bonds α to the hydroxyl group in ethanol and 2-propanol are approximately 93 kcal mol<sup>-1</sup> (389 kJ mol<sup>-1</sup>)<span class="cite-ref"><sup>[54,169]</sup></span> and 91 kcal mol<sup>-1</sup>,<span class="cite-ref"><sup>[54]</sup></span> respectively. Both values are slightly higher than the BDE of the O-H bond in a typical hydroperoxide ( ~ 88-90 kcal mol<sup>-1</sup> ).<span class="cite-ref"><sup>[170,171]</sup></span> This fact indicates that the autooxidation of typical primary and secondary alcohols, which is predominantly mediated by the peroxide radical, would be sluggish, since ethanol and 2-propanol can be viewed as the respective models for the oxidation of typical primary and secondary alcohols. The aldehydes/ketones formed can further oxidize through radical as well as non-radical-mediated oxidation pathways. Aldehydes usually undergo a radical-mediated oxidation pathway ultimately to give the corresponding carboxylic acids via the sequential intermediary of acyl radical and peracid.<span class="cite-ref"><sup>[172]</sup></span> A commonly seen pathway of the non-radical oxidation is the Baeyer-Villiger oxidation, which turns the acyclic ketones into esters, cyclic ketones into lactones, and aldehydes into carboxylic acids or formic esters.<span class="cite-ref"><sup>[136,173]</sup></span> The above pathways can be summarized in Scheme 3.43. The oxidation agent can be hydrogen peroxide or peracids; the latter can be viewed as activated forms of hydrogen peroxide. Different types of groups (R<sub>1</sub> and R<sub>2</sub>) in the Criegee intermediate have different propensities to migrate and, in general, certain types are more likely to migrate than others but the preference for migration can change depending upon the structure of the oxidation substrate and reaction conditions.<span class="cite-ref"><sup>[136]</sup></span> To simplify the discussion, the R<sub>2</sub> group is assumed to have a higher propensity to migrate in Scheme 3.43.</p>
</td><td>

<p>伯醇氧化生成醛，仲醇氧化生成酮。而此两者似乎都经历自由基机理。Williams 等人以电子自旋共振(EPR)研究了无定型态的含羟基药物分子的自然氧化，实验中发现并归属了两类自由基：碳自由基和过氧自由基。<span class="cite-ref"><sup>[168]</sup></span> 乙醇和异丙醇分子中，羟基 α 位的 C-H 键的离解能(bond dissociation energies, BDE)分别为 93 kcal mol<sup>-1</sup> (389 kJ mol<sup>-1</sup>) <span class="cite-ref"><sup>[54,169]</sup></span> 和 91 kcal mol<sup>-1</sup> <span class="cite-ref"><sup>[54]</sup></span>。这略高与过氧化物中 O-H 键的离解能（~ 88-90 kcal mol<sup>-1</sup>）。<span class="cite-ref"><sup>[170,171]</sup></span> 而乙醇和异丙醇可分别看做是典型的伯醇和仲醇，这意味着过氧自由基主导的醇的自然氧化反应往往是速度缓慢的。生成的醛/酮可被进一步氧化（包括自由基途径或非自由基途径）。会在自由基氧化中，醛往往会被最终氧化为羧酸，此间多会涉及酰基自由基和过氧酸。<span class="cite-ref"><sup>[172]</sup></span> 比较常见的非自由基氧化反应则是 Baeyer-Villiger 氧化：酮被氧化为酯；环酮成为内酯；醛生成羧酸或甲酸酯。<span class="cite-ref"><sup>[136,173]</sup></span> 上述反应总结于 Scheme 3.43。氧化剂可以是过氧化氢或过氧酸，而后者可看做是过氧化氢的活化形态。Criegee 中间体中，不同的基团(R<sub>1</sub> 和 R<sub>2</sub>)，迁移活性亦不同。一般来说，某特定类型的基团优先迁移，但也受底物结构和反应条件的制约。<span class="cite-ref"><sup>[136]</sup></span> 为了方便讨论，此处假定 R<sub>2</sub> 更容易迁移。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.43.png" alt="Scheme 3.43  " /><p class="caption"><span class="pic-ref">Scheme 3.43</span>  </p>
</div>
</td></tr>
<tr><td>

<p>It appears that there are not many examples of autooxidation of primary and secondary alcohol moieties in drug molecules; this could be attributed to the sluggishness of this type of autooxidation as discussed previously in this section. One notable example is the facile autooxidation of the 11β-hydroxyl group in certain corticosteroids when the latter compounds are present in a few solvated crystal forms.<span class="cite-ref"><sup>[174]</sup></span> For example, a solvated crystal form of hydrocortisone 21-tert-butylacetate was found to undergo spontaneous oxidation to yield the corresponding 11β-keto degradant (i.e. cortisone 21-tert-butylacetate, Scheme 3.44). When stored in ambient conditions for between 1 to 2 years; up to ~40% of this solvated form was oxidized at the 11β-hydroxyl position.</p>
</td><td>

<p>似乎伯醇或仲醇的自然氧化在药物分子并不多见，这是或许可归因于本章所探讨过的自然氧化反应在现实中进行的极其缓慢。一个值得注意的例子是，某些皮质类固醇药物的溶剂化物晶体中，11β-羟基可发生自然氧化。<span class="cite-ref"><sup>[174]</sup></span> 例如 hydrocortisone 21-tert-butylacetate 某一溶剂合物晶体，可自发氧化为相应的 11β-keto 化合物（例如，cortisone 21-tert-butylacetate，Scheme 3.44）。在空气中保存 1~2 年，将会有约 40% 的 11β-羟基被氧化。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.44.png" alt="Scheme 3.44  " /><p class="caption"><span class="pic-ref">Scheme 3.44</span>  </p>
</div>
</td></tr>
<tr><td>

<p>On the other hand, a non-solvated crystal form of the same compound was found to be completely stable under the same oxidative conditions. According to a study by Lin et al. who determined the structures of several crystal polymorphs, the reactivity toward the autooxidation in the solvated form can be attributed to a channel in the crystal through which molecular oxygen is capable of penetrating.<span class="cite-ref"><sup>[175]</sup></span> No chemical mechanism for the autooxidation was discussed in these studies. It can, nonetheless, be assumed that the autooxidation of the 11β-hydroxyl to 11β-keto proceeds via the free radical-mediated pathway as illustrated in Scheme 3.43.</p>
</td><td>

<p>但是，此化合物的无溶剂合晶体在同条件下却是非常稳定的。参考 Li 等人的所测定的多种晶型的晶体结构，溶剂合晶体明显容易发生自然氧化是因为此晶型中存在便于氧气分子渗入的通道。<span class="cite-ref"><sup>[175]</sup></span> 此研究中并未讨论化学反应机理。同时，也可将 11β-hydroxyl 自然氧化为 11β-keto 看做是经历了自由基机理，如 Scheme 3.43。</p>
</td></tr>
<tr><td>

<p>Another example can be found in the autooxidation of dexamethasone in an ophthalmic suspension. In several expired commercial batches, the 21-hydroxyl group of the corticosteroid, which is a primary alcohol, was oxidized to the 21-aldehyde group.<span class="cite-ref"><sup>[166]</sup></span> The dexamethasone 21-aldehyde formed underwent further oxidation to give a dexamethasone 17-formyloxy degradant, the process of which is most likely to be mediated through a Baeyer-Villiger oxidation of the 21-keto aldehyde moiety as shown in Scheme 3.45.</p>
</td><td>

<p>另一个例子是 dexamethasone 的眼用悬浊液。在数批过期制剂中发现，伯醇（21-羟基）被氧化为 21-醛。<span class="cite-ref"><sup>[166]</sup></span> 醛经历 Baeyer-Villiger 反应进一步氧化生成 dexamethasone 17-formyloxy-17-acid (Scheme 3.45)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.45.png" alt="Scheme 3.45  " /><p class="caption"><span class="pic-ref">Scheme 3.45</span>  </p>
</div>
</td></tr>
<tr><td>

<p>It is also worthwhile discussing the autooxidation of hydroxyl-bearing pharmaceutical excipients such as benzyl alcohol, polyethylene glycol (PEG), and polysorbate, owing to their wide application. Benzyl alcohol is commonly used as a preservative in pharmaceutical products at levels of 3-5%.<span class="cite-ref"><sup>[176]</sup></span> It is known that benzyl alcohol is susceptible to autooxidation, producing low level of benzaldehyde. Abend et al. found that the benzaldehyde formed can react with two moles of benzyl alcohol to give benzaldehyde dibenzyl acetal.<span class="cite-ref"><sup>[180]</sup></span></p>
</td><td>

<p>此外，含羟基的制剂辅料的自然氧化也是值得讨论的，比如广泛使用的苄醇、聚乙二醇(PEG)、聚山梨醇酯。一般在制剂中加入约 3-5% 的苄醇作为防腐剂。<span class="cite-ref"><sup>[176]</sup></span> 众所周知，苄醇容易发生自然氧化，删除少量的苯甲醛。Abend 等人发现，生成的苯甲醛可与两分子的苄醇反应生成苯甲醛苄基缩醛。<span class="cite-ref"><sup>[180]</sup></span></p>
</td></tr>
<tr><td>

<p>The acetal formed can then readily undergo alcohol exchange with a variety of hydroxyl-bearing excipients such as propylene glycol, resulting in the formation of interfering impurity peaks which could cause considerable analytical challenges during pharmaceutical development. This scenario is summarized in Scheme 3.46.</p>
</td><td>

<p>缩醛可以和各种含羟基的赋形剂（比如丙二醇）发生醇交换(alcohol exchange)生成干扰性杂质，这将对分析方法开发造成很大困难。此转化过程见 Scheme 3.45。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.46.png" alt="Scheme 3.46  " /><p class="caption"><span class="pic-ref">Scheme 3.46</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Polyethylene glycol (PEG) and polysorbate (in particular polysorbate 20 and 80, trade names: Tween 20 and 80) are non-ionic detergents and emulsifiers that are widely used in pharmaceutical formulations. The key structural moiety of both oligomers/polymers is polyethoxylated alcohol which can be represented by the formula R-(OCH<sub>2</sub>CH<sub>2</sub>)<sub>n</sub>-OH. It has long been known that PEG<span class="cite-ref"><sup>[181,182]</sup></span> and polysorbate183 are readily susceptible to free radical-mediated autooxidation during storage and handling under ambient conditions. This susceptibility can be attributed to the autooxidative degradation of the polyethoxylated alcohol moiety, during which process the terminal hydroxyl group, as well as the abundant ether functionality, can be readily oxidized to produce a range of aldehydes including formaldehyde, formic acid,<span class="cite-ref"><sup>[184]</sup></span> alkoxyl formate,<span class="cite-ref"><sup>[185]</sup></span> as well as numerous hydroperoxides including hydrogen peroxide.<span class="cite-ref"><sup>[41]</sup></span> Although the degradation mechanism has not been fully elucidated, the overall process can be described in Scheme 3.47, based on the published results.</p>
</td><td>

<p>聚乙二醇(PEG)和聚山梨醇酯(商品名为吐温，一般为吐温 20 或吐温 80)是非离子型去污剂和乳化剂，广泛用于药物制剂。两者的关键结构（齐聚物或聚合物）都是聚乙氧基化脂肪醇，通式为 R-(OCH<sub>2</sub>CH<sub>2</sub>)<sub>n</sub>-OH。已知 PEG<span class="cite-ref"><sup>[181,182]</sup></span> 和聚山梨醇酯在储存或暴漏于空气中时容易发生自由基介导的自然氧化。这是因为聚乙氧基化脂肪醇结构的自然氧化降解：端位的羟基和连接重复单元的醚键可迅速被氧化生成醛（包括甲醛、甲酸<span class="cite-ref"><sup>[184]</sup></span>、烷氧基甲酸酯<span class="cite-ref"><sup>[185]</sup></span>）和多种过氧化物（包括过氧化氢）<span class="cite-ref"><sup>[41]</sup></span>。但其降解机理尚未完全弄清，基于已发表的研究结果，整个降解过程总结为 Scheme 3.47。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.47.png" alt="Scheme 3.47  " /><p class="caption"><span class="pic-ref">Scheme 3.47</span>  </p>
</div>
</td></tr>
<tr><td>

<p>The above autooxidation is most likely to be catalyzed by redox active transition metal ions as discussed in Section 3.2.1. Generally speaking, two types of carbon-centered radicals would be formed: one that is α to the terminal hydroxyl group (radical A) and the other α to the ether functionality (radical B). Reaction of radicals A and B with molecular oxygen, respectively, would produce the corresponding peroxide radicals which in turn would abstract hydrogen atoms to give the peroxides. The peroxide formed from radical A is a Criegee intermediate<span class="cite-ref"><sup>[186,187]</sup></span> which can undergo two degradation pathways. The first pathway (a) would eliminate H<sub>2</sub>O<sub>2</sub> yielding an aldehyde, while the second pathway (b) would undergo a Baeyer-Villiger type of rearrangement, resulting in the formation of an α-alkoxyl formate. The peroxide formed from radical B could go through a carbon-carbon bond breakage to produce a formaldehyde and β-alkoxyl formate according to the mechanism proposed by Waterman et al.<span class="cite-ref"><sup>[50,184]</sup></span> Breakage of a similar carbon-carbon bond in analogous β-hydroxyl peroxides to produce α,ω-diketones<span class="cite-ref"><sup>[188]</sup></span> and dialdehydes<span class="cite-ref"><sup>[189]</sup></span> has been reported.</p>
</td><td>

<p>上述自然氧化很可能是受氧化还原活性的过渡金属离子催化的（详见小节 3.2.1）。一般来说，将会形成两种碳自由基：一个是端位羟基的 α 位(自由基 A)，另一个是醚的 α 位(自由基 B)。自由基 A 和 B 分别于氧分子反应产生相应的过氧自由基，随后夺氢生成过氧化物。由自由基 A 生成的过氧化物恰是 Criegee 中间体（Criegee 重排反应的中间体<span class="cite-ref"><sup>[186,187]</sup></span>），它有两种分解途径：(a) 发生消除反应，离去 H<sub>2</sub>O<sub>2</sub>，生成醛。(b) 发生类似于 Baeyer-Villiger 反应的重排，生成 α-烷氧基甲酸酯。而由自由基 B 生成的过氧化物会发生碳碳键断裂生成醛和 β-烷氧基甲酸酯，此机理由 Waterman 提出。<span class="cite-ref"><sup>[50,184]</sup></span> 另有报道类似的 β-羟基过氧化物可发生类似的碳碳键断裂，生成 α,ω-二酮<span class="cite-ref"><sup>[188]</sup></span>或二醛<span class="cite-ref"><sup>[189]</sup></span>。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.48.png" alt="Scheme 3.48  " /><p class="caption"><span class="pic-ref">Scheme 3.48</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the case of drug degradation caused by the oxidation of a ketone moiety, the autooxidation of tirilazad mesylate in acidic solutions is worth some discussion.<span class="cite-ref"><sup>[190]</sup></span> This drug substance is a 21-amino substituted steroid that is typically formulated in pH 3.0 aqueous solutions for maximaum solubility. Oxidation of the α-aminoketone moiety was found to be one of the two major degradation pathways of the drug substance, the process of which was postulated to begin with the addition of hydrogen peroxide to the ketone to form the Criegee intermediate. The latter species would then undergo a Grob fragmentation process<span class="cite-ref"><sup>[191]</sup></span> to yield a carboxylic acid and an iminium intermediate, according to a study performed by Wenkert et al.<span class="cite-ref"><sup>[192]</sup></span> Further oxidation of the iminium intermediate by a second molecule of hydrogen peroxide should give the formic amide, which ultimately would hydrolyze to release the amine degradant. Alternately, the iminium intermediate can be directly hydrolyzed to form the amine degradant. Although the latter possibility was not discussed by the original authors, it would not be surprising if the alternate hydrolysis is the major degradation pathway of the iminium intermediate, considering the fact that the study by Wenkert et al. was carried out in methanol solution,<span class="cite-ref"><sup>[192]</sup></span> while the autooxidation of tirilazad mesylate occurred in an entirely aqueous environment.<span class="cite-ref"><sup>[190]</sup></span> Furthermore, the authors' observation that the formic amide degradant was present in a very low quantity is consistent with the direct hydrolysis pathway. The complete degradation pathways are shown in Scheme 3.48.</p>
</td><td>

<p>转观酮的自然氧化，有必要讨论一下 tirilazad mesylate 在酸性溶液中自然氧化。<span class="cite-ref"><sup>[190]</sup></span> 此药物是一个 21 位氨基取代的类固醇，一般选用溶解性最佳的 pH 3.0 作为制剂条件。此化合物有两种主要降解途径，其一为 α-氨基酮的氧化：参考 Wenkert 等人的研究<span class="cite-ref"><sup>[192]</sup></span>，其反应过程应该是，过氧化物加成酮羰基，生成 Criegee 中间体，随后发生 Grob 裂解<span class="cite-ref"><sup>[191]</sup></span>生成羧酸和亚胺离子。亚胺离子受过氧化氢氧化成为甲酰胺，并最终水解为胺；或直接水解胺。虽然原作者并未讨论亚胺离子直接水解的可能性，但考虑到 Wenkert 的实验使用甲醇为溶剂<span class="cite-ref"><sup>[192]</sup></span>，而此次自然氧化研究则使用水为溶剂<span class="cite-ref"><sup>[190]</sup></span>，直接水解成为主要降解途径亦并不突兀。此外，作者仅观测到少量的甲酰胺，这说明了亚胺离子倾向于直接水解。整个降解机理见 Scheme 3.48。</p>
</td></tr>
<tr><td>

<p>Conjugated ketones can also be susceptible to oxidation via the Baeyer-Villiger mechanism. For example, steroids containing a cyclohexenone A-ring were found to degrade to the corresponding enol lactones, resulting from the net insertion of an oxygen into the six membered-ring.<span class="cite-ref"><sup>[193,194]</sup></span> Minor lactone epoxides were also observed (Scheme 3.49).</p>
</td><td>

<p>共轭酮还容易发生 Baeyer-Villiger 反应而被氧化。例如，含有环己酮 A环的类固醇发生 Baeyer-Villiger 氧化，氧原子插入到六元环内，降解为相应的烯醇内酯。<span class="cite-ref"><sup>[193,194]</sup></span> 同时还观测到少量环氧化物(Scheme 3.49)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.49.png" alt="Scheme 3.49  " /><p class="caption"><span class="pic-ref">Scheme 3.49</span>  </p>
</div>
</td></tr>
<tr><td>

<p>For such a conjugated system, generally the vinyl group preferentially migrates,<span class="cite-ref"><sup>[195]</sup></span> but exceptions have been reported.<span class="cite-ref"><sup>[196,197]</sup></span></p>
</td><td>

<p>在此类共轭体系中，往往会是烯丙基发生迁移<span class="cite-ref"><sup>[195]</sup></span>。但也有例外见于报道。<span class="cite-ref"><sup>[196,197]</sup></span></p>
</td></tr>
<tr><td>

<h3 id="oxidation-of-aromatic-rings-formation-of-phenols-polyphenols-and-quinones"><a href="#oxidation-of-aromatic-rings-formation-of-phenols-polyphenols-and-quinones">3.5.9 Oxidation of Aromatic Rings: Formation of Phenols, Polyphenols, and Quinones</a></h3>
</td><td>

<h3 id="芳香环的氧化生成酚多酚醌"><a href="#芳香环的氧化生成酚多酚醌">3.5.9 芳香环的氧化：生成酚、多酚、醌</a></h3>
</td></tr>
<tr><td>

<p>It has long been known that hydroxylation of unactivated aromatic rings such as monoalkyl-substituted phenyl rings is a very common oxidative degradation pathway in drug metabolism.<span class="cite-ref"><sup>[198]</sup></span> However, this pathway does not appear to be significant in the autooxidation of drugs, as only a few such cases have been reported in the literature. In principle, HO<sup>•</sup> radical generated by the Udenfriend and/or Fenton reactions is capable of hydroxylating an unactivated phenyl ring. Frequently, there are &quot;spots&quot; in a drug substance that are more reactive or susceptible to oxidation by HO<sup>•</sup> or its precursor in the Udenfriend degradation pathway, H<sub>2</sub>O<sub>2</sub>, which may explain why hydroxylation of unactivated phenyl rings is usually not a significant event in drug degradation.</p>
</td><td>

<p>人们很早就知道，在药物体内代谢中，不活泼的芳香环（比如单烷基取代的苯环）发生羟基化是很常见的降解途径。<span class="cite-ref"><sup>[198]</sup></span> 但是，此类反应在药物的自然氧化中却并不明显，仅有少数实例见诸报道。原则上，由 Udenfriend 反应或 Fenton 反应生成的 HO<sup>•</sup> 可与不活泼的苯环反应。而药物分子中往往存在某些位点更容易（或更不容易）被 HO<sup>•</sup> 或 H<sub>2</sub>O<sub>2</sub> 氧化。这或许能解释不活泼苯环的羟基化为何在药物降解中不那么显著。</p>
</td></tr>
<tr><td>

<p>Wu et al. reported the oxidative degradation study of a thrombin inhibitor, <strong>L-375,378</strong>, in tablet as well as intravenous (i.v.) solution dosage forms.<span class="cite-ref"><sup>[199]</sup></span></p>
</td><td>

<p>Wu 等人报道了凝血酶抑制剂 <strong>L-375,378</strong> 在片剂和静脉注射(i.v.)溶液制剂中的氧化降解。<span class="cite-ref"><sup>[199]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/F3.12.png" alt="Figure 3.12   L-375,378 的结构式，已标示出氧化位点。 Structure of L-375,378 showing various oxidation sites." /><p class="caption"><span class="pic-ref">Figure 3.12</span>   <strong>L-375,378</strong> 的结构式，已标示出氧化位点。<br /> Structure of <strong>L-375,378</strong> showing various oxidation sites.</p>
</div>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.50.png" alt="Scheme 3.50  " /><p class="caption"><span class="pic-ref">Scheme 3.50</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Major degradants 1 and 2 were also seen as the major degradation products in the forced degradation of <strong>L-375,378</strong> with H<sub>2</sub>O<sub>2</sub>, which is consistent with the degradation pathway proposed above. This pathway can be viewed as another example of autooxidation through an epoxide intermediate as discussed in Section 3.5.2; the presence of the phenylethylamino group could promote the formation of the expoxide intermediate as shown in Scheme 3.50.</p>
</td><td>

<p>降解产物 1 和 2 同时也是双氧水强制降解 <strong>L-375,378</strong> 时的主要降解产物。这上文提出的降解途径相符。此降解途径也可以看做是经历环氧化物中间体的自然氧化（小节 3.5.2）。苯乙基胺结构促进了环氧化物中间体的形成(Scheme 3.50)。</p>
</td></tr>
<tr><td>

<p>In contrast to the non-activated phenyl rings, activated phenyl rings such as hydroxyphenyl (phenol) and alkoxylphenyl derivatives are quite susceptible to autooxidation leading to the formation of polyhydroxylated products such as catechols. For example, the widely used bronchial smooth muscle relaxant, albuterol (salbutamol), contains a 2,4-dialkylphenol moiety which is susceptible to autooxidation at the 5-position under ambient storage condition to produce 5-hydroxyalbuterol.<span class="cite-ref"><sup>[200]</sup></span> Although no specific degradation mechanism was proposed by the original authors, this autooxidation is very likely to start from the formation of the oxygen-centered phenolic radical, which resonates with three carbon-centered radicals (Scheme 3.51).</p>
</td><td>

<p>与不活泼的苯环正相反，活化的苯环（比如苯酚、烷氧基苯）非常容易发生自然氧化反应生成多酚（比如，邻苯二酚）。例如，广泛使用的支气管平滑肌松弛剂 albuterol (salbutamol)，其结构中含有 2,4-二烷基酚结构，暴露于空气中存储时，它很容易发生自然氧化生成 5-hydroxyalbuterol。<span class="cite-ref"><sup>[200]</sup></span> 虽然原作者并未给出明确的降解机理，但显而易见，其自然氧化极有可能是形成了酚氧自由基，此自由基可与三种碳自由基极限式构成共振(Scheme 3.51)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.51.png" alt="Scheme 3.51  " /><p class="caption"><span class="pic-ref">Scheme 3.51</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Reaction of the 5-position radical, the least sterically hindered carbon-centered radical, with oxygen should lead to the 5-peroxyl intermediate. From the latter intermediate, two pathways are possible for the formation of 5-hydroxyalbuterol. In the presence of a reducing impurity, the peroxide intermediate can be directly reduced to the 5-hydroxy degradant (pathway a). Alternatively, the peroxide can give rise to the 5-oxy radical either through catalysis by transition metal ions or simply via thermolysis (pathway b).</p>
</td><td>

<p>自由基位于六元环的 5位时，空间位阻最小，此极限式与氧气反应生成 5-peroxyl 中间体。之后有两条途径生成 5-hydroxyalbuterol：若存在某些还原性物质则被直接还原(途径 a)；受过渡金属催化或热解生成 5-氧自由基(途径 b)。</p>
</td></tr>
<tr><td>

<p>The polyhydroxylated species are very electron-rich compounds owing to the strong electron-donating effect exerted by the multiple hydroxyl and similar groups (e.g. methoxy) and hence are strong reducing agents. In particular, drugs containing 1,2- and 1,4-dihydroxyphenyl moieties can undergo facile autooxidation to produce the 1,2- and 1,4-quinones, respectively, via the corresponding semi-quinone intermediates. Quinones are good Michael acceptors and as such they can react with nuclephiles to form 1,4-Michael adducts; these usually quickly further autooxidize to yield 4-substituted quinones. All the above degradation pathways are summarized below in Scheme 3.52 using epinephrine as an example.<span class="cite-ref"><sup>[201,202]</sup></span></p>
</td><td>

<p>多羟基化物因羟基或类似基团（比如，甲氧基）的强供电子作用而成为富电子化合物，同时也是强还原剂。一般来说，含有 1,2- 或 1,4- 二羟基苯基结构的药物极其容易发生自然氧化而生成相应的二醌，此过程将经历半醌中间体。醌是良好的 Michael 受体，故而可以和亲核试剂反应生成 1,4-Michael 加成产物；这将进一步氧化生成 4位取代的苯醌。以 epinephrine 为例，上述的降解途径总结于 Scheme 3.52。<span class="cite-ref"><sup>[201,202]</sup></span></p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.52.png" alt="Scheme 3.52  " /><p class="caption"><span class="pic-ref">Scheme 3.52</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the above case, the 1,2-quinone formed was attacked by the neighboring amino group to produce the Michael adduct which was further oxidized to yield the final substituted quinone. When a neighboring nucleophile like this is absent, the quinone formed can react with the starting material (catechol) to produce a semi-quinone intermediate.<span class="cite-ref"><sup>[203]</sup></span> In such cases, the autooxidation becomes a self-catalytic process, that is, quinone, the intermediary oxidative degradant, promotes the oxidation of catechol by participating in comproportionation. Consequently, such a catechol can undergo further facile autooxidation.</p>
</td><td>

<p>本例中，1,2-苯醌受到氨基进攻发生 Michael 加成，随后进一步氧化生成最终的取代苯醌。若分子内缺少类似的亲核基团，此苯醌可与 Epinephrine (邻苯二酚结构) 反应生成半醌。<span class="cite-ref"><sup>[203]</sup></span> 如此则自然氧化可构成自催化：苯醌作为氧化剂，与邻苯二酚发生归中反应，促进氧化进行。故此，邻苯二酚会发生更进一步的自然氧化。</p>
</td></tr>
<tr><td>

<p>In another case of activated aromatic ring oxidation, Auclair and Paoletti found that the antitumor drug 9-hydroxyellipticine undergoes facile autooxidation in alkaline aqueous solutions producing, respectively, the corresponding quinone imine (9-oxoellipticine), a dimer of 9-hydroxyellipticine, and hydrogen peroxide (Scheme 3.53).<span class="cite-ref"><sup>[204]</sup></span> EPR experiments suggested that the autooxidation process involves the initial formation of a free radical of the drug and O<sub>2</sub><sup>-•</sup>. Both species could undergo their own disproportionation to yield hydrogen peroxide and the quinone imine 9-oxoellipticine, respectively. The latter compound is capable of reacting with the drug 9-hydroxyellipticine itself to form the dimer.</p>
</td><td>

<p>这还有一个活化的芳香环被氧化的例子，Auclair 和 Paoletti 发现，抗癌药 9-hydroxyellipticine 在碱性水溶液中可发生自然氧化生成相应的苯醌亚胺(9-oxoellipticine)、二聚体和过氧化氢(Scheme 3.53)。<span class="cite-ref"><sup>[204]</sup></span> EPR 实验显示，此自然氧化过程涉及此药物分子的自由基和 O<sub>2</sub><sup>-•</sup> 的形成。这两种自由基分别发生相应的歧化反应生成过氧化氢和醌亚胺 9-oxoellipticine。后者可与 9-hydroxyellipticine 反应生成二聚体。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.53.png" alt="Scheme 3.53  " /><p class="caption"><span class="pic-ref">Scheme 3.53</span>  </p>
</div>
</td></tr>
<tr><td>

<p>This autooxidation mechanism is somewhat different from that shown in Scheme 3.52 in that in the current case molecular oxygen acts &quot;directly&quot; as the oxidant (via catalysis through trace redox transition metal ions), while in Scheme 3.52 O<sub>2</sub><sup>-•</sup> was proposed as the oxidant. In the current case, the transition metal ions may directly chelate with the drug 9-hydroxyellipticine. Hence, this may be a Udenfriend type of autooxidation in which the oxidation substrate (the drug) serves as both the chelator and the reducing agent. This hypothesis by the current author is consistent with the observation made by Auclair and Paoletti<span class="cite-ref"><sup>[204]</sup></span> that use of EDTA dramatically decreased the autooxidation of the drug, because EDTA should disrupt the complex formed between the drug, transition metal ions, and oxygen. The reactive species, that is, O<sub>2</sub><sup>-•</sup>, the drug free radical, and the quinone, formed in the autooxidation process, as illustrated in Scheme 3.53 are potentially cytotoxic and their formation has been hypothesized to be responsible for the activity of the antitumor drug.</p>
</td><td>

<p>此自然氧化机理与 Scheme 3.52 略有不同。在 Scheme 3.52 中 O<sub>2</sub><sup>-•</sup> 才能作为是氧化剂，但本例中，氧分子“直接”作为氧化剂（受痕量的氧化还原活性的过渡金属离子催化），且过渡金属离子有可能直接与药物分子络合。因此，这可能是另一个底物既充当络合剂，又充当还原剂的 Udenfriend 型自然氧化反应。笔者的这一假设与 Auclair 和 Paoletti 观测到的实验结果相符<span class="cite-ref"><sup>[204]</sup></span>：在体系中加入 EDTA 可明显减少药物分子的自然氧化，这是因为 EDTA 可扰乱药物分子、过渡金属离子、氧分子之间的络合。自然氧化过程中生成的活性物类（即 O<sub>2</sub><sup>-•</sup>、药物分子的自由基、苯醌）皆列于 Scheme 3.53，它们都是具有强烈的细胞毒性。据猜测此药物的抗肿瘤活性与这些毒性分子有关。</p>
</td></tr>
<tr><td>

<p>Drugs like morphine and those related to it, for example, naloxone, nalbuphine, and oxymorphone,<span class="cite-ref"><sup>[205]</sup></span> contain an ortho-alkyoxyphenolic moiety that can undergo autooxidative carbon-carbon coupling to yield primarily the respective 2,2'-dimers. In the case of morphine, the 2,2'-morphine dimer is also called pseudomorphine. Autooxidative dimerization most probably starts from the formation of the phenolic radical which can be resonated at the 2- and 4-positions of the aromatic ring. The 2-phenolic radical can attack a morphine molecule to give the dimer radical which should readily autooxidize, leading to the formation of the dimer. The mechanism postulated<span class="cite-ref"><sup>[206]</sup></span> is illustrated in Scheme 3.54 using morphine as the example.</p>
</td><td>

<p>Morphine 及其相关药物（比如 naloxone、nalbuphine、和 oxymorphone<span class="cite-ref"><sup>[205]</sup></span>），含有邻烷氧基苯酚结构，因自然氧化可发生 C-C 偶联生成 2,2'-dimer。以 morphine 为例，2,2'-morphine dimer 又名 pseudomorphine。此二聚反应可能是先生成酚氧自由基，随后自由基共振到苯环的 2 位或 4 位，2-phenolic radical 进攻另一个 morphine 完成碳碳偶联，二聚体的自由基经氧化最终生成 pseudomorphine。此机理<span class="cite-ref"><sup>[206]</sup></span>见 Scheme 3.54。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.54.png" alt="Scheme 3.54  " /><p class="caption"><span class="pic-ref">Scheme 3.54</span>  </p>
</div>
</td></tr>
<tr><td>

<h3 id="oxidation-of-heterocyclic-aromatic-rings"><a href="#oxidation-of-heterocyclic-aromatic-rings">3.5.10 Oxidation of Heterocyclic Aromatic Rings</a></h3>
</td><td>

<h3 id="芳香杂环的氧化"><a href="#芳香杂环的氧化">3.5.10 芳香杂环的氧化</a></h3>
</td></tr>
<tr><td>

<p>Heterocyclic aromatic rings, such as imidazole, indole, pyridine, and their analogs including polycyclic rings that contain fused heterocyclic rings, are common structural features in various drug molecules. Examples of small molecule drugs that contain an imidazole ring include several azole antifungal drugs, clotrimazole, econazole, ketoconazole, isoconazole, and miconazole. For protein and peptide drugs, the imidazole-containing histidine residue is a key component that is frequently involved in metal ion binding of these drugs. As a result, the histidine residue of protein and peptide drugs becomes susceptible to a transition metal ion-mediated autooxidation, in particular cupric ion. According to the results obtained by several groups, the oxidative degradant resulting from the transition metal ion-mediated process, via the Fenton chemistry, is 2-oxohistidine.<span class="cite-ref"><sup>[207-209]</sup></span> In this metal ion-mediated oxidation, HO<sup>•</sup> was postulated as the reactive oxygen species that attacks the imidazole ring at the 2-position (pathway a, Scheme 3.55).<span class="cite-ref"><sup>[210]</sup></span> Nevertheless, a more recent EPR study of the histidine oxidation indicated that the hydroxyl radical predominantly attacks the 4-position to form the 5-position radical rather than the 2-position radical (pathway b).<span class="cite-ref"><sup>[211]</sup></span> To reconcile with the ultimate formation of 2-oxohistidine residue, Schöneich<span class="cite-ref"><sup>[210]</sup></span> proposed a dehydrated radical intermediate produced from the 5-position radical (pathway b1). Alternatively, the current author postulates another possible pathway leading to the formation of the 2-oxohistidine (pathway b). All the proposed mechanisms are summarized in Scheme 3.55.</p>
</td><td>

<p>芳香杂环（比如咪唑、吲哚、吡啶以及其他含有杂原子的稠环）常见于各种药物分子。含有咪唑环的小分子药物如唑类抗真菌药：clotrimazole、econazole、ketoconazole、isoconazole 以及 miconazole。在蛋白或多肽类药物中，含有咪唑环的组氨酸残基可参与对金属离子的络合，而这往往是药物发挥作用关键。但这也使得组氨酸残基容易受过渡金属离子的影响而发生自然氧化，且铜离子尤为明显。由多个研究小组的成果可知，在过渡金属离子催化的 Fenton 体系中，组氨酸残基将被氧化为 2-氧代组氨酸。<span class="cite-ref"><sup>[207-209]</sup></span> 据推测，此时 HO<sup>•</sup> 是最主要的活性氧类，它进攻咪唑环的 2 位(途径 a，Scheme 3.55)。<span class="cite-ref"><sup>[210]</sup></span> 但是不久前，有人使用 EPR 研究了组氨酸的氧化过程，却发现羟基自由基主要进攻咪唑环的 4 位而在形成 5 位形成自由基（并非是之前推测的 2 位），此即途径 b。<span class="cite-ref"><sup>[211]</sup></span> 为了解释最终产物 2-氧代组氨酸的形成，Schöneich<span class="cite-ref"><sup>[210]</sup></span> 提出 5 位自由基脱水生成另一个自由基中间体(途径 b1)。另外，笔者构造了另一条可能途径最终形成 2-氧代组氨酸(途径 b)。以上所讨论的反应机理见 Scheme 3.55。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.55.png" alt="Scheme 3.55  " /><p class="caption"><span class="pic-ref">Scheme 3.55</span>  </p>
</div>
</td></tr>
<tr><td>

<p>How pathway b could be preferred over pathway a may be rationalized based on the fact that the 5-position radical is a tertiary radical which should be more stable than the alternative, secondary radicals. On the other hand, pathway b1 seems quite unlikely according to the isotope experiment with H<sub>2</sub><sup>18</sup>O in which no <sup>18</sup>O was found to be incorporated into 2-oxohistidine.<span class="cite-ref"><sup>[210]</sup></span> This result mitigates strongly against pathway b1 where rehydration of the dehydrated radical intermediate by water should lead to the incorporation of the water oxygen.</p>
</td><td>

<p>但为什么组氨酸的氧化会经历途径 b 而不是途径 a 呢？这是因为 5 位是叔碳，所形成的自由基更稳定。另一方面，以同位素标记的 H<sub>2</sub><sup>18</sup>O 进行实验，却没能在 2-氧代组氨酸中发现 <sup>18</sup>O。<span class="cite-ref"><sup>[210]</sup></span> 这表明途径 b1 并不存在，因为脱水而成的自由基中间体会再次水解，则必然会有一部分终产物的氧原子来自于水。</p>
</td></tr>
<tr><td>

<p>For the azole antifungal drugs mentioned above, the imidazole ring is linked to the rest of the drug molecule through the 1-nitrogen position. As such, oxidation of the imidazole ring would probably be different from that of the histidine residue. A search of the literature revealed no results for the autooxidation of the imidazole ring in these drugs, except a stress study of miconazole by the free radical initiator, azobisisobutyronitrile (AIBN),<span class="cite-ref"><sup>[212]</sup></span> which is frequently used to simulate autooxidative degradation of drug substances. In this study, the 1-nitrogen substituted imidazole ring was oxidized to 2,4,5-trioxoimidazole. The original authors hypothesized that triplet molecular oxygen reacts with the imidazole ring in the same way as singlet oxygen based on the fact that the latter is known to react with imidazole to produce unstable dioxygenated intermediates.<span class="cite-ref"><sup>[213-215]</sup></span> Nevertheless, the formation of 2,4,5-triox-oimidazole may also be explained by a mechanism that only involves triplet molecular oxygen, as illustrated in Scheme 3.56, that does not violate the spin conservation rule.</p>
</td><td>

<p>上文提过的另一个唑类抗真菌药 miconazole，分子中存在 1-N 取代的咪唑环。此咪唑环的氧化行为不同于组氨酸残基。笔者检索文献并未发现对此分子中咪唑环的自然氧化研究，但有一例使用自由基引发剂 AIBN (azobisisobutyronitrile，常用于模拟药物分子的自然氧化降解) 进行的强制降解研究。<span class="cite-ref"><sup>[212]</sup></span> 此强制降解中，咪唑环被氧化为 2,4,5-trioxoimidazole。已知单线态氧分子与咪唑环反应可生产不稳定的 dioxygenated 中间体<span class="cite-ref"><sup>[213-215]</sup></span>，原作者推测三线态氧分子可发生同样的反应。但是，Scheme 3.56 所示的机理也足以解释 2,4,5-trioxoimidazole 的生成，只涉及三线态氧分子且不违背自旋守恒规则。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.56.png" alt="Scheme 3.56  " /><p class="caption"><span class="pic-ref">Scheme 3.56</span>  </p>
</div>
</td></tr>
<tr><td>

<p>In the above mechanism, the abundant AIBN-generated peroxyl radicals can attack either the 2-position (pathway a) or the 5-position (pathway b) to yield radicals that are more stable than the alternatives owing to the resonances shown in Scheme 3.56. Reaction of the radicals with triplet molecular oxygen would give the corresponding peroxyl radicals, which in turn can produce the peroxides by abstracting hydrogens from a general hydrogen donor (RH). Elimination of a water and alcohol from the two peroxides in pathways a and b should form the same 2,5-dioxoimidazolyl intermediate. Further oxidation of the latter by another AIBN-generated peroxyl radical can lead to the formation of 2,4,5-trioxoimidazole degradant.</p>
</td><td>

<p>上述机理中，由 AIBN 产生大量的过氧自由基可进攻咪唑环的 2 位(途径 a)或 5 位(途径 b)，生成两种相对稳定的自由基中间体，随后与三线态氧分子反应生成相应的过氧自由基，过氧自由基夺氢成为相应的过氧化物。过氧化物消除脱去水或醇生成 2,5-dioxoimidazolyl 中间体。此物进一步被过氧自由基氧化则生成最终降解产物 2,4,5-trioxoimidazole。</p>
</td></tr>
<tr><td>

<p>The indole ring is a common structural feature present in many natural products,<span class="cite-ref"><sup>[216]</sup></span> which are important sources of new drugs.<span class="cite-ref"><sup>[217]</sup></span> It is also a side chain of the most hydrophobic protein amino acid, tryptophan. Its UV absorption at 280 nm is mostly responsible for the characteristic absorption of proteins and peptides that contain tryptophan residues. The most significant oxidation of indole ring is the oxidation of its fused pyrrole ring; an example was discussed in the case of indomethacin oxidation in Section 3.5.2. Owing to its importance in proteins and peptides, there will be further discussion of its autooxidative degradation pathways in Chapter 7, Chemical Degradation of Biological Drugs.</p>
</td><td>

<p>吲哚环是众多天然产物分子中的常见结构<span class="cite-ref"><sup>[217]</sup></span>，而天然产物是新药分子的重要来源<span class="cite-ref"><sup>[217]</sup></span>。色氨酸侧链中即含有吲哚环，其紫外最大吸收波长为 280 nm，恰是含色氨酸残基的多肽和蛋白的最佳检测波长。吲哚的氧化反应中，最重要的莫过于稠和吡咯环被氧化，小节 3.5.2 中曾以 indomethacin 为例讨论过此反应。由于吲哚环频频出现蛋白和多肽中，我们将在第七章进一步讨论其自然氧化降解。</p>
</td></tr>
<tr><td>

<p>Towards the low end of oxidizability is the pyridine moiety which is an electron-deficient species, a weak base, as well as a weak nucleophile. Consequently, the pyridine moiety is usually stable under autooxiative conditions where electrophilic oxygen transfer is the predominant oxidation mechanism.<span class="cite-ref"><sup>[66]</sup></span> On the other hand, N-oxidation of the pyridine moiety is commonly observed in drug metabolism.<span class="cite-ref"><sup>[218]</sup></span></p>
</td><td>

<p>最不容易被氧化的是吡啶环，这是一个缺电子物类、弱碱和若亲核剂。因此，吡啶环在亲电氧转移机理为主导的自然氧化条件下一般能保持稳定。<span class="cite-ref"><sup>[66]</sup></span> 但是，在药物体内代谢中时常发现吡啶的 N-氧化物。<span class="cite-ref"><sup>[218]</sup></span></p>
</td></tr>
<tr><td>

<h3 id="miscellaneous-oxidative-degradations"><a href="#miscellaneous-oxidative-degradations">3.5.11 Miscellaneous Oxidative Degradations</a></h3>
</td><td>

<h3 id="其他氧化降解"><a href="#其他氧化降解">3.5.11 其他氧化降解</a></h3>
</td></tr>
<tr><td>

<p>Boron is an element that people have tried to incorporate into drug molecules but with few successes. Bortezomib, a potent first-in-class dipeptidyl boronic acid 20S proteasome inhibitor used for the treatment of relapsed multiple myeloma, is perhaps the only approved boron-containing drug. During a preformulation study, Wu et al. found that the drug underwent facile auto-oxidation in solutions to produce two major hydroxyl degradants along with a couple of secondary and tertiary degradants.<span class="cite-ref"><sup>[219]</sup></span> The two major degradants are epimers with respect to each other with the configuration difference lying in the hydroxylated sites. To explain the formation of the two epimeric diastereomers, the original authors hypothesized the existence of an acylated Schiff base that would result from the dehydration of hydroxyl degradant 1. The epimerization could then occur by rehydration of the Schiff base to yield hydroxyl degradant 2 (Scheme 3.57).</p>
</td><td>

<p>人们曾尝试把硼原子引入到药物分子中，但成功案例不多。二肽基硼酸 Bortezomib 是首创(first in class)的 20S 蛋白酶抑制剂，是目前唯一获批的含硼药物，用于治疗恶性多发性骨髓瘤。在其制剂处方前研究中，Wu 等人发现此药物在溶液中很容易发生自然氧化，产生两个羟基化的主要降解产物，并伴随有数个次级降解产物。<span class="cite-ref"><sup>[219]</sup></span> 这两个主要降解产物是一对差向异构体——羟基所在的那个碳原子的手性不同。为了解释这对异构体的生成，原作者猜想 hydroxyl degradant 1 将形成相应的酰化希夫碱，进而水解发生消旋化生成 hydroxyl degradant 2 (Scheme 3.57)。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.57.png" alt="Scheme 3.57  " /><p class="caption"><span class="pic-ref">Scheme 3.57</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Nevertheless, during the drug metabolism study, Labutti et al. observed two intermediary peroxyl degradants which can be converted into the two hydroxyl degradants.<span class="cite-ref"><sup>[220]</sup></span> Moreover, these authors also made two additional important observations: first, the oxygen atoms of the hydroxyl degradants originate solely from molecular oxygen rather than water through the use of <sup>18</sup>O<sub>2</sub> and H<sub>2</sub><sup>18</sup>O, respectively, which rules out the Schiff base mechanism as the cause for racemization. Second, stressing a solution of bortezomib with FeSO<sub>4</sub> generated a degradation profile that is very similar to those obtained from the in vivo drug metabolism study. These results strongly indicate that the chemical degradation and metabolic transformation of bortezomib should share very similar degradation pathways. Hence, a chemical degradation mechanism that is more consistent with all the experimental observations is illustrated in Scheme 3.58.</p>
</td><td>

<p>此外，在药物代谢研究中，Latutti 等人观测到了两个过氧化物中间体，可进一步转化为上述的两个羟基化降解产物。<span class="cite-ref"><sup>[220]</sup></span> 而且，他们还观察到了两个重要事实：其一，羟基化降解产物的氧原子来自于氧气而非水分子（分别以同位素标记的 <sup>18</sup>O<sub>2</sub> 和 H<sub>2</sub><sup>18</sup>O 实验可知）。这排除了希夫碱机理。其二，以 FeSO<sub>4</sub> 强制降解 bortezomib 溶液所得结果与体内代谢情况非常相似。这足以说明其化学降解与体内代谢经历了相似的机理。于是提出了 Scheme 3.58 所示的化学降解机理，以解释实验中观测到的事实。</p>
</td></tr>
<tr><td colspan=2>

<div class="figure">
<img src="png/S3.58.png" alt="Scheme 3.58  " /><p class="caption"><span class="pic-ref">Scheme 3.58</span>  </p>
</div>
</td></tr>
<tr><td>

<p>Since a fair amount of hydroxyl degradant 2 was observed in both cases by the two research groups, the predominant oxidative degradation of bortezomib may be rationalized by a free radical mechanism that can explain the substantial racemization that occurred. It has been reported that alkylboronates can undergo non-radical (or polar) as well as radical-mediated oxidative pathways.<span class="cite-ref"><sup>[221]</sup></span> In the non-radical (polar) pathway of the oxidative degradation, the alkyl group retains its original configuration. In the radical mechanism presented in Scheme 3.58, the H<sup>•</sup> donor, RH, can be bortezomib, in which case radical 1 may be generated again. It is also worth noting that during the preformulation study performed by Wu et al.,<span class="cite-ref"><sup>[219]</sup></span> use of either ascorbic acid or EDTA in the formulation was found to promote the oxidation of bortezomib. This may be another case in which the Udenfriend chemistry (see Section 3.2.1) may be responsible for initiating the autooxidative degradation.</p>
</td><td>

<p>两研究小组都观测到了相当多的 hydroxyl degradant 2，而自由基降解机理能够对消旋化给出一个合理的解释。曾有报道，烷基硼酸酯可也发生非自由基介导（或称极性）的氧化反应。<span class="cite-ref"><sup>[221]</sup></span> 在非自由基（极性）氧化降解机理中，烷基可保持原始构型。而在 Scheme 3.58 所示的自由基机理下，bortezomib 可充当所谓的 H<sup>•</sup> 供体——RH，这将生成另一个 radical 1。另外，在 Wu 等人的制剂处方前研究中<span class="cite-ref"><sup>[219]</sup></span>，值得一提的是，加入维生素C 或 EDTA 可促进 bortezomib 的氧化。这很有可能又是 Udenfriend 反应参与引发了自然氧化降解。</p>
</td></tr>
</table>

<h2 id="references"><a href="#references">References</a></h2>
<ol style="list-style-type: decimal">
<li><p>W. O. Lumberg (ed), Autoxidation and Antioxidants John Wiley &amp; Sons, 1961, Vol. 1, p. 2.</p></li>
<li><p>R. Willstatter and A. Stoll, Justus Liebigs Ann. Chem., 1911, 387, 317.</p></li>
<li><p>R. Willstatter and A. Stoll, in Untersuchungen uber Chlorophyll, Springer, Berlin, 1913.</p></li>
<li><p>D. M. Miller, G. R. Buettner and S. D. Aust, Free Rad. Biol. Med., 1990, 8, 95.</p></li>
<li><p>M. K. Eberhardt, Reactive Oxygen Metabolites, CRC Press, Boca Raton, FL, 2001.</p></li>
<li><p>B.-Z. Zhu and G.-Q. Shan, Chem. Res. Toxicol., 2009, 22, 969.</p></li>
<li><p>J. S. Edmonds, M. Morita, P. Turner, B. W. Skelton and A. H. White, Steroids, 2006, 71, 34.</p></li>
<li><p>P. A. Harmon, S. Biffar, S. M. Pitzenberger and R. A. Reed, Pharm. Res., 2005, 22, 1716.</p></li>
<li><p>H. J. H. Fenton, J. Chem. Soc. Trans., 1894, 65, 899.</p></li>
<li><p>F. Haber and J. J. Weiss, Proc. R.. Soc. London, Ser. A, 1934, 147, 332.</p></li>
<li><p>S. Udenfriend, C. T. Clark, J. Axelrod and B. B. Brodie, J. Biol. Chem., 1954, 208, 731.</p></li>
<li><p>C. A. Reed, in The Biological Chemistry of Iron, ed. H.B. Dunford, D. Dolphin, K.M. Raymond and L. Sieker, D. Reidel, Dordrecht, 1982, pp. 25-42.</p></li>
<li><p>G. Schwarzenbach and J. Heller, Helv. Chim. Acta, 1951, 34, 576.</p></li>
<li><p>W. H. Koppenol and J. Butler, Adv. Free Radical Biol. Med., 1985, 1, 91.</p></li>
<li><p>C. Walling, Acc. Chem. Res., 1975, 8, 125.</p></li>
<li><p>P. A. MacFaul, D. D. M. Wayner and K. U. Ingold, Acc. Chem. Res., 1998, 31, 159.</p></li>
<li><p>S. Goldstein, D. Meyerstein and G. Czapski, Free Rad. Biol. Med., 1993, 15, 435.</p></li>
<li><p>D. T. Sawyer, A. Sobkowiak and T. Matsushita, Acc. Chem. Res., 1996, 29, 409.</p></li>
<li><p>O. Pestovsky, S. Stoian, E. L. Bominaar, X. Shan, E. Miinck, L. J. Que and A. Bakac, Angew. Chem., Int. Ed., 2005, 44, 6871.</p></li>
<li><p>J. England, M. Martinho, E. R. Farquhar, J. R. Frisch, E. L. Bominaar, E. Miinck and L. J. Que, Angew. Chem., Int. Ed., 2009, 48, 3622.</p></li>
<li><p>A. Bakac, Inorg. Chem., 2010, 49, 3584.</p></li>
<li><p>J. T. Groves, Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 3569.</p></li>
<li><p>J. Rittle and M. T. Green, Science, 2010, 330, 933.</p></li>
<li><p>E. R. Stadtman, Free Rad. Biol. Med., 1990, 9, 315.</p></li>
<li><p>E. R. Stadtman and C. N. Oliver, J. Biol. Chem., 1991, 266, 2005.</p></li>
<li><p>D. R. Dufield, G. S. Wilson, R. S. Glass and C. Schoneich, J. Pharm. Sci., 2004, 93, 1122.</p></li>
<li><p>M. K. Eberhardt, Trends Org. Chem., 1995, 5, 115.</p></li>
<li><p>H. Kasai and S. Nishimura, Nucleic Acids Res., 1984, 12, 2137.</p></li>
<li><p>M. Li, S. Carlson, J. A. Kinzer and H. J. Perpall, Biochem. Biophys. Res. Commun., 2003, 312, 316.</p></li>
<li><p>M. D. Engelmann, R. T. Bobier, T. Hiatt and I. F. Cheng, BioMetals, 2003, 16, 519.</p></li>
<li><p>G. Schwarzenbach and J. Heller, Helv. Chim. Acta, 1951, 34, 1889.</p></li>
<li><p>E. Bottari and G. Anderegg, Helv. Chim. Acta, 1967, 50, 2349.</p></li>
<li><p>G. H. Buettner, Arch. Biochem. Biophys., 1993, 300, 535.</p></li>
<li><p>K. M. Ko, P. K. Yick, M. K. T. Poon and S. P. Ip, Mol. Cell. Biochem., 1994, 141, 65.</p></li>
<li><p>A. Aguiar and A. Ferraz, Chemosphere, 2007, 66, 947.</p></li>
<li><p>J. E. Biaglow and A. V. Kachur, Radiat. Res., 1997, 148, 181.</p></li>
<li><p>B. W. Alderman, A. E. Ratliff and J. I. Wirgau, Inorg. Chim. Acta, 2009, 362, 1787.</p></li>
<li><p>J. Hong, E. Lee, J. C. Carter, J. A. Masse and D. A. Oksanen, Pharm. Dev. Technol., 2004, 9, 171.</p></li>
<li><p>J. W. McGinity, T. R. Patel and A. H. Naqvi, Drug Dev. Commun., 1976, 2, 505.</p></li>
<li><p>T. Huang, M. E. Garceau and P. Gao, J. Pharm. Biomed. Anal., 2003, 31, 1203.</p></li>
<li><p>W. R. Wasylaschuk, P. A. Harmon, G. Wagner, A. B. Harman, A. C. Templeton, H. Xu and R. A. Reed, J. Pharm. Sci., 2007, 96, 106.</p></li>
<li><p>V. L. Antonovskii and S. L. Khursan, Russ. Chem. Rev., 2003, 72, 939.</p></li>
<li><p>P. A. Harmon, K. Kosuda, E. Nelson, M. Mowery and R. A. Reed, J. Pharm. Sci., 2006, 95, 2014.</p></li>
<li><p>A. J. Bard and L. R. Faulkner in Electrochemical Methods-Fundamentals and Applications, John Wiley and Sons, New York, 1980.</p></li>
<li><p>W. H. Koppenol, FEBS Lett., 1990, 264, 165.</p></li>
<li><p>K. U. Ingold, Acc. Chem. Res., 1969, 2, 1.</p></li>
<li><p>J. A. Howard and K. U. Ingold, Can. J. Chem., 1967, 45, 793.</p></li>
<li><p>G. A. Russell, J. Am. Chem. Soc., 1957, 79, 3871.</p></li>
<li><p>G. W. Burton and K. U. Ingold, J. Am. Chem. Soc, 1981, 103, 6472.</p></li>
<li><p>K. C. Waterman, R. C. Adami, K. M. Alsante, J. Hong, M. S. Landis, F. Lombardo and C. J. Roberts, Pharm. Dev. Technol., 2002, 7, 1.</p></li>
<li><p>Handbook of Chemistry and Physics, 75th edn, CRC Press, Boca Raton, FL. 1995.</p></li>
<li><p>M. Jonsson, J. Phys. Chem., 1996, 100, 6814.</p></li>
<li><p>N. Sebbar, J. W. Bozzelli and H. Bockhorn, J. Phys. Chem. A, 2004, 108, 8353.</p></li>
<li><p>D. F. McMillen and D. M. Golden, Ann. Rev. Phys. Chem., 1982, 33, 493.</p></li>
<li><p>F. R. Cruickshank and S. W. Benson, J. Phys. Chem., 1969, 73, 733.</p></li>
<li><p>F. R. Cruickshank and S. W. Benson, Int. J. Chem. Kinet., 1969, 1, 381.</p></li>
<li><p>W. S. Nip and G. Paraskevopoulos, J. Chem. Phys., 1979, 71, 2170.</p></li>
<li><p>G. A. Russell, J. Am. Chem. Soc., 1955, 78, 1035.</p></li>
<li><p>C. J. Norton, F. L. Dormish, M. J. Reuter, N. F. Seppi and P. M. Beazley, Ind. Eng. Chem. Prod. Res. Dev., 1972, 11, 27.</p></li>
<li><p>M. A. Freyaldenhoven, P. A. Lehman, T. J. Franz, R. V. Lloyd and V. M. Samokyszyn, Chem. Res. Toxicol., 1998, 11, 102.</p></li>
<li><p>A. M. Arafat, S. K. Mathew, S. O. Akintobi and A. A. Zavitsas, Helv. Chim. Acta, 2006, 89, 2226.</p></li>
<li><p>C. W. Capp and E. G. E. Hawkins, J. Chem. Soc., 1955, 4106.</p></li>
<li><p>C. J. Toney, F. E. Friedli and P. J. Frank, J. Am. Oil Chem. Soc., 1994, 71, 793.</p></li>
<li><p>W. R. Thiel, Coord. Chem. Rev, 2003, 245, 95.</p></li>
<li><p>Z. Zhu and J. H. Espenson, J. Org. Chem., 1995, 60, 1326.</p></li>
<li><p>W. J. Szczepek, B. Kosmacinska, A. Bielejewska, W. Luniewski, M. Skarzynski and D. Rozmarynowskaa, J. Pharm. Biomed. Anal., 2007, 43, 1682.</p></li>
<li><p>A. L. Freed, H. E. Strohmeyer, M. Mahjour, V. Sadineni, D. L. Reid and C. A. Kingsmill, Int. J. Pharm., 2008, 357, 180.</p></li>
<li><p>R. S. Drago, A.L.M.L. Mateus and D. Patton, J. Org. Chem., 1996, 61, 5693.</p></li>
<li><p>G. B. Payne, P. H. Deming and P. H. Williams, J. Org. Chem., 1961, 26, 659.</p></li>
<li><p>Y. Sawaki and Y. Ogata, Bull. Chem. Soc. Japan, 1981, 54, 793.</p></li>
<li><p>S. W. Hovorka, M. J. Hageman and C. Schoneich, Pharm. Res., 2002, 19, 538.</p></li>
<li><p>B. Balagam and D. E. Richardson, Inorg. Chem., 2008, 47, 1173.</p></li>
<li><p>R. D. Bach, M. N. Glukhovtsev and C. Gonzalez, J. Am. Chem. Soc., 1998, 120, 9902.</p></li>
<li><p>H. Yao and D. E. Richardson, J. Am. Chem. Soc., 2000, 122, 3220.</p></li>
<li><p>E. Weitz and A. Scheffer, Chem. Ber., 1921, 54, 2327.</p></li>
<li><p>C. A. Bunton and G. J. Minkoff, J. Chem. Soc., 1949, 665.</p></li>
<li><p>G. Sosnovsky and E. H. Zaret in Organic Peroxides, ed. D. Swern, Wiley, New York, 1970, Vol. 1, p. 517.</p></li>
<li><p>I. P. Skibida and A. M. Sakharov, Catal. Today, 1996, 187.</p></li>
<li><p>G. A. Russell, J. Am. Chem. Soc., 1954, 76, 1595.</p></li>
<li><p>G. A. Russell and A. G. Bemis, J. Am. Chem. Soc., 1966, 88, 5491.</p></li>
<li><p>D. H. R. Barton and D. W. Jones, J. Chem. Soc., 1965, 3563.</p></li>
<li><p>M. Li, B. Chen, S. Monteiro and A. M. Rustum, Tetrahedron Lett., 2009, 50, 4575.</p></li>
<li><p>J. Hansen and H. Bundgaard, Int. J. Pharmaceut., 1980, 6, 307.</p></li>
<li><p>E. V. Bejan, E. Font-Sanchis and J. C. Scaiano, Org. Lett., 2001, 3, 4059.</p></li>
<li><p>C. Pan, F. Liu, Q. Ji, W. Wang, D. Drinkwater and R. Vivilecchia, J. Pharm. Biomed. Anal., 2006, 40, 581.</p></li>
<li><p>A. Mohan, M. Hariharan, E. Vikraman, G. Subbaiah, B. R. Venkataraman and D. Saravanan, J. Pharm. Biomed. Anal., 2008, 47, 183.</p></li>
<li><p>United States Pharmacopoeia 30, United States Pharmacopoeial Convention, Rockville, MD, p. 1802.</p></li>
<li><p>B. Proksa, J. Pharm. Biomed. Anal., 1999, 20, 179.</p></li>
<li><p>H. Farsam, S. Eiger, J. Lameh, A. Rezvani, B. W. Gibson and W. Sadee, Pharm. Res., 1990, 7, 1205.</p></li>
<li><p>S. S. Kelly, P. M. Glynn, S. J. Madden and D. H. Grayson, J. Pharm. Sci., 2003, 92, 485.</p></li>
<li><p>A. M. Kamel, K. S. Zandi and W. W. Massefski, J. Pharm. Biomed. Anal., 2003, 31, 1211.</p></li>
<li><p>J. D. Stong, J. V. Pivnichny, H. Mrozik and F. S. Waksmunski, J. Pharm. Sci, 1992, 81, 1000.</p></li>
<li><p>Q. Wang, J. D. Stong, P. Demontigny, J. M. Ballard, J. S. Murphy, J.-S. K. Shim and A. J. Faulkner, J. Pharm. Sci., 1996, 85, 446.</p></li>
<li><p>S. Javernik, S. Kreft, B. Strukelj and F. Vrecer, Pharmazie, 2001, 56, 738.</p></li>
<li><p>G. B. Smith, L. DiMichele, L. F. Colwell, Jr., G. C. Dezeny, A. W. Douglas, R. A. Reamer and T. R. Verhoeven, Tetrahedron Lett., 1993, 49, 4447.</p></li>
<li><p>R. S. Tomar, T. J. Joseph, A. S. R. Murthy, D. V. Yadav, G. Subbaiah and K.V.S.R. Krishna Reddy, J. Pharm. Biomed. Anal., 2004, 36, 231.</p></li>
<li><p>M. L. Huang, A. V. Peer, W. Robert, R. D. Coster, D. V. M. J. Heykants and A. A. I. Jonkman, Pharm. Drug Dispos., 1993, 54, 257.</p></li>
<li><p>R. P. Enever, A. Li Wan and Po and E. Shotton, J. Pharm. Sci., 1979, 68, 169.</p></li>
<li><p>R. P. Enever, A. Li Wan, Po, B. J. Millard and E. Shotton, J. Pharm. Sci., 1975, 64, 1497.</p></li>
<li><p>G. Callen, M. S. Chorghade, E. C. Lee, P. G. Nilsen, H. Petersen and A. Rustum, Heterocycles, 1994, 39, 293.</p></li>
<li><p>X. Zhang and C. S. Foote, J. Am. Chem. Soc., 1993, 115, 8867.</p></li>
<li><p>M. Li, B. Conrad, R. G. Maus, S. M. Pitzenberger, R. Subramanian, X. Fang, J. A. Kinzer and H. J. Perpall, Tetrahedron Lett., 2005, 46, 3533.</p></li>
<li><p>G. Boccardi, C. Deleuze, M. Gachon, G. Palmisano and J. P. Vergnaud, J. Pharm. Sci., 1992, 81, 183.</p></li>
<li><p>http://drugbank.wishartlab.com (last accessed March 2011).</p></li>
<li><p>X. Wang, M. Li and A. M. Rustum, Rapid Commun. Mass Spectrom., 2010, 24, 2805.</p></li>
<li><p>X. Li, F. E. Blondino, M. Hindle, W. H. Soine and P. R. Byron, Int. J. Pharm., 2005, 303, 113.</p></li>
<li><p>S. B. Karki and J. P. Dinnocenzo, Xenobiotica, 1995, 25, 711.</p></li>
<li><p>S. B. Karki, J. P. Dinnocenzo, J. P. Jones and K. R. Korzekwa, J. Am. Chem. Soc., 1995, 117, 3657.</p></li>
<li><p>H. Matsumoto, M. Fukumoto and A. Ogamo, Jpn. J. Forensic Toxicol., 1998, 16, 164.</p></li>
<li><p>Z. Z. Zhao, X.-Z. Qin, A. Wu and Y. Yuan, J. Pharm. Sci., 2004, 93, 1957.</p></li>
<li><p>J. Dong, S. B. Karki, M. Parikh, J. C. Riggs and L. Huang, Drug Dev. Ind. Pharm., posted online on January 23, 2012.</p></li>
<li><p>R. Ramanathan, A.-D. Su, N. Alvarz, N. Blumenkrantz, S. K. Chowdhury and J. E. Patrick, Anal. Chem., 2000, 72, 1352.</p></li>
<li><p>S. K. Chowdhury and K. B. Alton, Anal. Chem., 2005, 77, 3676.</p></li>
<li><p>A. C. Cope, T. T. Foster and P. H. Towle, J. Am. Chem. Soc., 1949, 71, 3929.</p></li>
<li><p>S. Ma, S. K. Chowdhury and K. B. Alton, Anal. Chem., 2005, 77, 3676.</p></li>
<li><p>J. Meisenhemimer, Chem. Ber., 1919, 52, 1667.</p></li>
<li><p>H. E. De La Mare, J. Org. Chem, 1960, 25, 2114.</p></li>
<li><p>A. L. J. Beckwith, P. H. Eichinger, B. A. Mooney and R. H. Prager, Aust. J. Chem, 1983, 36, 719.</p></li>
<li><p>Z. Foldi, T. Foldi and A. Foldi, Chem. Ind, 1955, 1297.</p></li>
<li><p>J.-E. Belgaied and H. Trabelsi, J. Pharm. Biomed. Anal., 2003, 33, 991.</p></li>
<li><p>W. D. Emmons, J. Am. Chem. Soc., 1957, 79, 5528.</p></li>
<li><p>K. M. Ibne-Rasa and J. O. Edwards, J. Am. Chem. Soc., 1962, 84, 763.</p></li>
<li><p>B. C. Challis and A. R. Butler, in The Chemistry of the Amino Group, ed. S. Patai, Wiley and Sons, London, 1968, pp. 320-338.</p></li>
<li><p>D. H. Rosenblatt and E. P. Burrows, in Supplement F: The Chemistry of Amino, Nitroso, and Nitro Compounds and Their Derivatives, Part 2, ed. S. Patai, Wiley and Sons, Chichester, 1982, pp. 1085-1149.</p></li>
<li><p>J. D. Fields and P. J. Kropp, J. Org. Chem., 2000, 65, 5937.</p></li>
<li><p>C. Zonta, E. Cazzola, M. Mba and G. Licinia, Adv. Synth. Catal., 2008, 350, 2503.</p></li>
<li><p>H.-C. Shi and Y. Li, J. Mol. Catal. A: Chem., 2007, 271, 32.</p></li>
<li><p>J. Zheng and A. M. Rustum, J. Pharm. Biomed. Anal., 2010, 51, 146.</p></li>
<li><p>J. F. W. Keana, Chem. Rev, 1978, 78, 37.</p></li>
<li><p>W. Jahnke, S. Rudisser and M. Zurini, J. Am. Chem. Soc., 2001, 123, 3149.</p></li>
<li><p>M. F. Semmelhack, C. S. Chou and D. A. Cortes, J. Am. Chem. Soc., 1983, 105, 4492.</p></li>
<li><p>M. R. Leanna, T. J. Sowin and H. E. Morton, Tetrahedron Lett., 1992, 33, 5029.</p></li>
<li><p>S. K. Malhotra, J. J. Hostynek and A. F. Lundin, J. Am. Chem. Soc., 1968, 90, 6565.</p></li>
<li><p>G. Modena and P. E. Todesco, J. Chem. Soc., 1962, 4920.</p></li>
<li><p>J. W. Chu and B. L. Trout, J. Am. Chem. Soc., 2004, 126, 900.</p></li>
<li><p>G. R. Krow in Organic Reactions, Vol. 43, ed. L. A. Paquette et al., John Wiley &amp; Sons, 1993, pp. 251-798.</p></li>
<li><p>F. Di Furia and G. Modena, Pure Appl. Chem., 1982, 54, 1853.</p></li>
<li><p>B. L. Miller, T. D. Williams and C. Schoneich, J. Am. Chem., Soc., 1996, 118, 11014.</p></li>
<li><p>B. L. Miller, K. Kuczera and C. Schoneich, J. Am. Chem. Soc., 1998, 120, 3345.</p></li>
<li><p>K.-D. Asmus, in Sulfur-centered Reactive Intermediates in Chemistry and Biology, ed. C. Chatgilialoglu and K.-D. Asmus, NATO ASI Series 197, Plenum Press, New York, 1990, pp. 155-172.</p></li>
<li><p>C. Schoneich, A. Aced and K.-D. Asmus, J. Am. Chem. Soc., 1993, 115, 11376.</p></li>
<li><p>K. Bobrowski, G. L. Hug, D. Pogocki, B. Marciniak and C. Schoneich, J. Phys. Chem. B, 2007, 111, 9608.</p></li>
<li><p>C. Schoneich, Biochim. Biophys. Acta, 2005, 1703, 111.</p></li>
<li><p>I. Fourre and J. Bergès, J. Phys. Chem. A, 2004, 108, 898.</p></li>
<li><p>M. L. Huang and A. Rauk, J. Phys. Chem. A, 2004, 108, 6222.</p></li>
<li><p>P. Brunelle and A. Rauk, J. Phys. Chem. A, 2004, 108, 11032.</p></li>
<li><p>C. Schoneich, Arch. Biochem. Biophys., 2002, 397, 370.</p></li>
<li><p>A. Rauk, D. A. Armstrong and D. P. Fairlie, J. Am. Chem. Soc., 2000, 122, 9761.</p></li>
<li><p>R. S. Glass, Top. Curr. Chem., 1999, 205, 1.</p></li>
<li><p>E. D. Nelson, P. A. Harmon, R. C. Szymanik, M. G. Teresk, L. Li, R. A. Seburg and R. A. Reed, J. Pharm. Sci., 2006, 95, 1527.</p></li>
<li><p>M. M. Al Omari, R. M. Zoubi, E. I. Hasan, T. Z. Khader and A. A. Badwan, J. Pharm. Biomed. Anal., 2007, 45, 465.</p></li>
<li><p>H. A. Rosenberg, J. T. Dougherty, D. Mayron and J. G. Baldinus, Am. J. Hosp. Pharm., 1980, 37, 390.</p></li>
<li><p>S. E. Walker, T. W. Paton, T. M. Fabian, C. C. Liu and P. E. Coates, Am. J. Hosp. Pharm., 1981, 38, 881.</p></li>
<li><p>P. M. G. Bavin, A. Post and J. E. Zarembo, in Analytical Profiles of Drug Substances, ed. K. Florey, Academic Press, Orlando, 1984, Vol. 13, pp. 127-183.</p></li>
<li><p>G. W. Mihaly, O. H. Drummer, A. Marshall, R. A. Smallwood and W. J. Louis, J. Pharm. Sci., 1980, 69, 1155.</p></li>
<li><p>N. Beaulieu, P. A. Lacroix, R. W. Sears and E. G. Lovering, J. Pharm. Sci, 1988, 77, 889.</p></li>
<li><p>M. J. Puz, B. A. Johnson and B. J. Murphy, Pharm. Dev. Technol., 2005, 1, 115.</p></li>
<li><p>V. Caplar, S. Rendic, F. Kajfez, H. Hofman, J. Kuftinec and N. Blazevic, Acta Pharm. Jugosl., 1982, 32, 125.</p></li>
<li><p>K. A. Connors, G. L. Amidon and V. J. Stella, Chemical Stability of Pharmaceuticals: A handbook for pharmacists. John Wiley &amp; Sons, New York, 2nd edn, 1986.</p></li>
<li><p>M. Benrahmoune, P. Therond and Z. Abedinzadeh, Free Radicals Biol. Med, 2000, 29, 775.</p></li>
<li><p>L. Gu, H.-S. Chiang and A. Becker, Int. J. Pharm., 1988, 41, 95.</p></li>
<li><p>B. Mao, A. Abrahim, Z. Ge, D. K. Ellison, R. Hartman, S. V. Prabhu, R. A. Reamer and J. Wyvratt, J. Pharm. Biomed. Anal., 2002, 28, 1101.</p></li>
<li><p>L. R. Reddy and E. J. Corey, Tetrahedron Lett., 2005, 46, 927.</p></li>
<li><p>H. Bundgaard and J. Hansen, Arch. Pharm. Chemi., Sci. Ed., 1980, 8, 187.</p></li>
<li><p>T. Chulski and A. A. Forist, J. Am. Pharm. Assoc., Sci. Ed., 1958, 47, 553.</p></li>
<li><p>R. E. Conrow, G. W. Dillow, L. Bian, L Xue, O. Papadopoulou, J. K. Baker and B. S. Scott, J. Org. Chem., 2002, 67, 6835.</p></li>
<li><p>D. V. C. Awang, A. Vincent and F. Matsui, J. Pharm. Sci., 1973, 62, 1673.</p></li>
<li><p>H. E. Williams, V. C. Loades, M. Claybourn and D. M. Murphy, Anal. Chem, 2006, 78, 604.</p></li>
<li><p>Handbook of Chemistry and Physics, ed. R. C. Weast, 61st edn, CRC Press, Boca Raton, FL, pp. 1980-1981.</p></li>
<li><p>S. J. Blanksby, T. M. Ramond, G. E. Davico, M. R. Nimlos, S. Kato, V. M. Bierbaum, W. C. Lineberger, G. B. Ellison and M. Okumura, J. Am. Chem. Soc., 2001, 123, 9585.</p></li>
<li><p>D. M. Johnson and L. C. Gu, in Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick and J.C. Boylan, Marcel Dekker, New York, 1988, Volume 1, pp. 415-449.</p></li>
<li><p>R. A. Sheldon and J. K. Kochi, Metal-Catalyzed Oxidations of Organic Compounds, Academic Press, New York, 1981, p. 359.</p></li>
<li><p>M. Matsumoto, H. Kobayashi and Y. Hotta, J. Org. Chem., 1984, 49, 4740.</p></li>
<li><p>G. Brenner, F. E. Roberts, A. Hoinowski, J. Budavari, B. Powell, D. Hinkley and E. Schoenewaldt, Angew. Chem., Int. Ed., 1969, 8, 975.</p></li>
<li><p>C.-T. Lin, P. Perrier, G. G. Clay, P. A. Sutton and S. R. Byrn, J. Org. Chem, 1982, 47, 2978.</p></li>
<li><p>A. H. Kibbe, Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 2000, pp. 41-43.</p></li>
<li><p>S. Korcek, J. H. B. Chenier, J. A. Howard and K. U. Ingold, Can. J. Chem, 1972, 50, 2285.</p></li>
<li><p>A. B. Levina, S. R. Trusov and Z. Obshchei, Khimii, 1990, 60, 1932.</p></li>
<li><p>V. R. Choudhary, P. A. Chaudhari and V. S. Narkhede, Catal. Commun., 2003, 4, 171.</p></li>
<li><p>A. M. Abend, L. Chung, R. Todd Bibart, M. Brooks and D. G. McCollum, J. Pharm. Biomed. Anal., 2004, 34, 957.</p></li>
<li><p>W. G. Llyod, J. Chem. Eng. Data, 1961, 6, 541.</p></li>
<li><p>R. Hamburger, E. Azaz and M. Donbrow, Pharm. Acta Helv., 1975, 50, 10.</p></li>
<li><p>M. Donbrow, E. Azaz and A. Pillersdorf, J. Pharm. Sci., 1978, 67, 1676.</p></li>
<li><p>K. C. Waterman, W. B. Arikpo, M. B. Fergione, T. W. Graul, B. A. Johnson, B. C. MacDonald, M. C. Roy and R. J. Timpano, J. Pharm. Sci, 2008, 97, 1499.</p></li>
<li><p>M. Bergh, L. P. Shao, K. Magnusson, E. Gafvert, J. L. G. Nilsson and A.-T. Karlberg, J. Pharm. Sci., 1999, 88, 483.</p></li>
<li><p>B. Plesnicar, in The Chemistry of Peroxides, ed. S. Patai, John Wiley &amp; Sons, Chichester, UK, 1983, p. 559.</p></li>
<li><p>M. Renz and B. Meunier, Eur. J. Org. Chem., 1999, 737.</p></li>
<li><p>A. Nishinaga, K. Rindo and T. Matsuura, Synthesis, 1986, 1038.</p></li>
<li><p>H. Yuasa, M. Matsuno and H. Imai, Eur. Pat. Appl., EP 103099 A2, 1984.</p></li>
<li><p>B. G. Snider, T. A. Runge, P. E. Fagerness, R. H. Robins and B. D. Kaluzny, Int. J. Pharm., 1990, 66, 63.</p></li>
<li><p>C. A. Grob, Angew. Chem., Int. Ed., 1969, 8, 535.</p></li>
<li><p>D. Wenkert, K. M. Eliasson and D. Rudisill, J. Chem. Soc., Chem. Commun., 1983, 392.</p></li>
<li><p>E. Caspi, Y. W. Chang and R. I. Dorfman, J. Med. Pharm. Chem., 1962, 5, 714.</p></li>
<li><p>J. T. Pinhey and K. Schaffner, Aust. J. Chem., 1968, 21, 1873.</p></li>
<li><p>G. A. Krafft and J. A. Katzenellenbogen, J. Am. Chem. Soc., 1981, 103, 5459.</p></li>
<li><p>M. S. Ahmad and A. R. Siddiqi, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 1978, 16, 963.</p></li>
<li><p>S. D. Levine, J. Org. Chem, 1966, 31, 3189.</p></li>
<li><p>B. B. Brodie, J. R. Gillette and B. N. La Du, Annu. Rev. Biochem., 1958, 27, 427.</p></li>
<li><p>Y. Wu, X. Chen, L. Gier, O. Almarsson, D. Ostovic and A. E. Loper, J. Pharm. Biomed. Anal., 1999, 20, 471.</p></li>
<li><p>N. K. Yee, L. J. Nummy and G. P. Roth, Bioorg. Med. Chem. Lett., 1996, 6, 2279.</p></li>
<li><p>H. P. Misra and I. Fridovich, J. Biol. Chem., 1972, 247, 3170.</p></li>
<li><p>K. A. Connors, G. L. Amidon and V. J. Stella, Chemical Stability of Pharmaceuticals: A handbook for pharmacists, John Wiley &amp; Sons, New York, 2nd edn, 1986, pp. 438-447.</p></li>
<li><p>G. Li, H. Zhang, F. Sader, N. Vadhavkar and D. Njus, Biochemistry, 2007, 46, 6978.</p></li>
<li><p>C. Auclair and C. Paoletti, J. Med. Chem., 1981, 24, 289.</p></li>
<li><p>M. A. Quarry, D. S. Sebastian and F. Diana, J. Pharm. Biomed. Anal., 2002, 30, 99.</p></li>
<li><p>S.-Y. Yeh and J. L. Lach, J. Pharm. Sci., 1961, 50, 35.</p></li>
<li><p>K. Uchida and S. Kawakishi, Arch. Biochem. Biophys., 1990, 283, 20.</p></li>
<li><p>K. Uchida and S. Kawakishi, FEBS Lett., 1993, 332, 208.</p></li>
<li><p>F. Zhao, E. Ghezzo-Schoneich, G. I. Aced, J. Hong, T. Milby and C. Schoneich, J. Biol. Chem., 1997, 272, 9019.</p></li>
<li><p>C. Schöneich, J. Pharm. Biomed. Anal., 2000, 21, 1093.</p></li>
<li><p>G. Lassmann, L. A. Eriksson, F. Himo, F. Lendzian and W. Lubitz, J. Phys. Chem. A, 1999, 103, 1283.</p></li>
<li><p>A. R. Oyler, R. E. Naldi, K. L. Facchine, D. J. Burinsky, M. H. Cozine, R. Dunphy, J. D. Alves-Santana and M. L. Cotter, Tetrahedron, 1991, 47, 6549.</p></li>
<li><p>H. Wasserman, K. Stiller and M. Floyd, Tetrahedron Lett., 1968, 29, 3277.</p></li>
<li><p>H. H. Wasserman, M. S. Wolff, K. Stiller, I. Saito and J. E. Pickett, Tetrahedron, 1981, 37, 191.</p></li>
<li><p>H.-S. Ryang and C. S. Foote, J. Am. Chem. Soc., 1979, 101, 6683.</p></li>
<li><p>J. W. Blunt, B. R. Copp, W.-P. Hu, M. H. G. Munro, P. T. Northcote and M. R. Prinsep, Nat. Prod. Rep., 2007, 24, 31.</p></li>
<li><p>D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461.</p></li>
<li><p>P. Kulanthaivel, R. J. Barbuch, R. S. Davidson, P. Yi, G. A. Rener, E. L. Mattiuz, C. E. Hadden, L. A. Goodwin and W. J. Ehlhardt, Drug Metab. Dispos., 2004, 32, 966.</p></li>
<li><p>S. Wu, W. Waugh and V. J. Stella, J. Pharm. Sci, 2000, 89, 758.</p></li>
<li><p>J. Labutti, I. Parsons, R. Huang, G. Miwa, L.-S. Gan and J. S. Daniels, Chem. Res. Toxicol., 2006, 19, 539.</p></li>
<li><p>C. Cadot, P. I. Dalko and J. Cossy, J. Org. Chem., 2002, 67, 719.</p></li>
</ol>
<hr />
<!-- vim:se ft=markdown: -->


</body>
</html>
